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Is polymerase chain reaction fully reliable? A critical review in the light of published evidence from the golden decades of molecular genetics

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Abstract

Soon after its introduction in 1987, Polymerase Chain Reaction (PCR) has become a cycle-based technique widely employed in diagnostic medical devices and in forensic science with the intention of amplifying genetic information. The achievement of a sufficient de-gree of fidelity is mandatory for reliability in applications; however, PCR prescribes that each of its cycles must include a heating subprocess at 95°C or more, denominated DNA denaturation, which may entail a risk of disruption of the DNA molecule. Understanding that the conditio sine qua non for the fidelity of PCR is to prevent such a dis-ruption, a review is presented of the historical literature of the years 1950-1960 elucidating the effects of heating on possible DNA fragmentation. The main conclusion of this review is that the body of examined evidence consistently and redundantly confirms such disrup-tion when DNA is heated at temperatures above 90°C, even for one minute. This appears to contradict the paradigm of PCR fidelity and leads the authors to raise the concern that PCR may amplify information, but, at least for long sequences, in an unreliable way. This should open a discussion on what the PCR paradigm could signify in various fields in which PCR is used.
Preprint for
bioR
iv 2021, 10, x. https://doi.org/10.3390/xxxxx
Scientific Review Article
1
Is polymerase chain reaction fully reliable? A critical review
2
in the light of published evidence from the golden decades
3
of molecular genetics
4
Roberto Serpieri 1 and Fabio Franchi 2,*
5
1 University of Campania Luigi Vanvitelli, Aversa, Italy; roberto.serpieri@unicampania.it
6
2 Società Scientifica per il Principio di Precauzione (SSPP), Roma, Italy. Former Dirigente Medico (M.D)
7
in Infectious Disease Ward, specialized in “Infectious Diseases” and “Hygiene and Preventive Medi-
8
cine”, Trieste, Italy
9
* Correspondence: roberto.serpieri@unicampania.it; Tel.: +39 339 1991634
10
Simple Summary: Soon after its introduction in 1987, Polymerase Chain Reaction (PCR) has
11
become a cycle-based technique widely employed in diagnostic medical devices and in foren-
12
sic science with the intention of amplifying genetic information. The achievement of a suffi-
13
cient degree of fidelity is mandatory for reliability in applications; however, PCR prescribes
14
that each of its cycles must include a heating subprocess at 95°C or more, denominated DNA
15
denaturation, which may entail a risk of disruption of the DNA molecule.
16
Understanding that the conditio sine qua non for the fidelity of PCR is to prevent such a disrup-
17
tion, a review is presented of the historical literature of the years 1950-1960 elucidating the
18
effects of heating on possible DNA fragmentation. The main conclusion of this review is that
19
the body of examined evidence consistently and redundantly confirms such disruption when
20
DNA is heated at temperatures above 90°C, even for one minute. This appears to contradict
21
the paradigm of PCR fidelity and leads the authors to raise the concern that PCR may amplify
22
information, but, at least for long sequences, in an unreliable way. This should open a discus-
23
sion on what the PCR paradigm could signify in various fields in which PCR is used.
24
Abstract: The purpose of this review is to verify the reliability of the PCR test in amplifying
25
the genetic information contained in the samples to be tested. The fundamentals of PCR in-
26
clude, in each of its amplification cycles, a phase of denaturation of DNA by heating, which
27
according to the proponents allows the two chains of the molecule to be separated, followed
28
by a cooling phase intended to allow the formation of two equal molecular sequences of DNA.
29
The repetition of the procedure for several times would allow to obtain huge quantities of the
30
same sequence, thus allowing easier detection and analysis of a desired sequence. The possi-
31
bility of fragmentation could however compromise the final result. While this is excluded by
32
the inventor, scientific literature on the matter is far from being unanimous.
33
Tracing back to the seminal studies on DNA heating, proof of longitudinal sequence-breaking
34
random thermal fragmentation of DNA molecules is found. To ascertain this phenomenon,
35
three main measurement methodologies have been applied on native and heated DNA by in-
36
dependent group of researchers: sedimentation rate, viscometry, and light scattering. The ex-
37
amined experimental studies agree all but a few exceptions in finding important alterations
38
of DNA even after one heating-cooling cycle: it would seem amplified DNA molecules could
39
not maintain their original structure.
40
The real significance of PCR is questioned, and a thorough revaluation suggested on this mat-
41
ter and its consequences.
42
Keywords: PCR; thermal depolymerization; amplification fidelity; diagnostic reliability; ge-
43
netic information integrity.
44
Citation: Serpieri, R.; Franchi, F.;
Is polymerase chain reaction fully
reliable? A critical review in the light
of published evidence from the
golden decades of molecular gene-
tics . BioR
ive 2021, XX, x.
https://doi.org/XX.XXXX/xxxxx
Received: 08/11/2021
Accepted: DD/MM/YYYY
Published: DD/MM/YYYY
Copyright: © 2021 by the authors.
Submitted for possible open access
publication under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(https://creativecommons.org/license
s/by/4.0/).
bioR
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Sommario in italiano: Poco dopo la sua introduzione nel 1987, la Polymerase Chain Reaction
45
(PCR), una tecnica basata sulla reiterazione di procedimenti ciclici di manipolazione del mate-
46
riale genetico, ha trovato largo impiego, in ambito medico diagnostico e nelle scienze forensi,
47
in dispositivi impiegati dagli utenti con l’intento di amplificare l’informazione genetica conte-
48
nuta in campioni di materiale. Il raggiungimento di un grado di fedeltà di amplificazione suf-
49
ficiente è fondamentale per garantire affidabilità nelle applicazioni; tuttavia, la PCR prescrive
50
che in ciascuno dei suoi cicli sia incluso un processo di riscaldamento a temperature uguali o
51
superiori a 95°C, denominato denaturazione del DNA, che può comportare il rischio di di-
52
struggere le molecole di DNA.
53
Procedendo dalla considerazione che la conditio sine qua non affinché il requisito di fedeltà della
54
PCR sia garantito è che sia scongiurata una distruzione caotica della molecola oggetto di am-
55
plificazione, viene qui presentata una revisione della letteratura storica a cavallo degli anni
56
1950-1960 allo scopo di chiarire gli effetti del riscaldamento sulla possibile frammentazione del
57
DNA. La conclusione principale di questa revisione è che il corpo complessivo di evidenze
58
esaminate conferma consistentemente e ridondantemente una siffatta distruzione quando il
59
DNA è riscaldato a temperature superiori ai 90°C anche per un solo minuto. Questa conclu-
60
sione appare contraddire il paradigma della fedeltà della PCR e induce gli autori a sollevare la
61
preoccupazione che la PCR possa amplificare l’informazione ma, almeno per sequenze di lun-
62
ghezza maggiore, in un modo non affidabile e non controllabile. Questa conclusione dovrebbe
63
sollecitare una discussione sul reale significato del paradigma della PCR nei vari campi in cui
64
questa tecnica è impiegata.
65
66
67
68
69
1. Introduction
70
71
As narrated by himself in an autobiographic note [1], in 1983 Kary Mullis
72
was struck by the idea of a procedure for synthesizing repeatedly Deoxyribo-
73
Nucleic Acid (DNA) in vitro, which he named Polymerase Chain Reaction
74
(PCR). Beginning with a single target molecule of DNA, he envisaged he
75
could find DNA fragments and “generate 100 billion similar molecules in an af-
76
ternoon”. In 1985 he applied for a patent for PCR [2], then finished and substi-
77
tuted it by a subsequent 1987 application [3]. He described such procedure
78
also in a scientific record [4] in the format of an experimental method. The
79
Authors report [3,4] that PCR is based upon the reiteration of a reaction cycle
80
which includes, in each cycle, a heating subprocess referred to as denaturing
81
or denaturation. In the “Claims” section of the patent application [3], denatur-
82
ation was so mentioned: wherein said nucleic acid is double stranded and its
83
strands are separated by denaturing. Hereafter, we will refer to such heating
84
subprocess intended for DNA strands separation as the denaturation claim
85
or, more briefly, as DNA denaturation.
86
The presence of a DNA heating step, in a conceivable theoretical scheme
87
for nucleic acid amplification and sequencing, turns out to be a critical point
88
for the sake of the fidelity of such an amplification procedure against the risk
89
of thermal depolymerization, or disaggregation, of the DNA molecule. This
90
could lead to a consequential loss of information in the sequence of nucleo-
91
tides, due to the random breakage mechanisms similar to those explaining
92
the molecular weight decay observed under the effect of gamma-rays on
93
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DNA, described for instance by Cox et al. (1955) [5], and reported also for
94
heating by many authors. However, concerning DNA stability to heating,
95
there can be found relevant discrepancies between more recent studies deal-
96
ing with the exploitation of DNA heating above 80°C for nucleic acid ampli-
97
fication purposes, and the results in the original early studies devoted to the
98
analysis of the disruptive effects of heat on DNA. Such discrepancies are
99
briefly sketched hereafter.
100
Innis et al. in 1988 [6], cited by Kary Mullis in 1990 [1], reported a remark-
101
able fidelity of PCR by heating. It is stated indeed [6] that with PCR segments
102
of single-copy genomic DNA can be amplified >10 million-fold with very high speci-
103
ficity and fidelity” and (referring to a modified version of PCR therein pro-
104
posed) that the fidelity of PCR […] is quite respectable (approximately one mistake
105
in 4000-5000 base pairs sequenced after 35 cycles of PCR and cloning of the prod-
106
ucts)”. They write The mixture was heated to 90°C for 3 min, incubated at 42°C
107
for 20 min, cooled to room temperature”, but no specific considerations or refer-
108
ences are reported concerning the problem of a thermal depolymerization.
109
Similarly, in [3,4] it is reported the possibility to customarily perform heating
110
steps from a temperature of about 80° up to temperatures as high as 100°C
111
and even 105°C [3].
112
Conversely, the very early pioneering studies of historical relevance
113
which were entirely dedicated to the physical, chemical and biological effects
114
on DNA by several physical agents [7,8], provided compelling evidence that
115
heating above 81°, even for few minutes, determines a sharp thermal DNA
116
degradation. The observed degradation also consists in strands fragmenta-
117
tion into shorter molecular segments involving deterministically unpredicta-
118
ble sequence-breaking fractures of linkages along a single polynucleotide
119
chain. This phenomenon was early shown by several authors. Among others,
120
Shooter et al. carefully experimentally examined it [9], while Applequist in
121
1961 well described its bond scission kinetics by a random degradation model
122
[10] achieving significant predictivity of several degradation processes. Also,
123
the biological implications concomitant to DNA fragmentation with loss of
124
biological activity were early shown as a first macroscopic effect by Zamen-
125
hof et al. (1953) [8].
126
It is worth anticipating that, in this respect, a watershed publication deal-
127
ing with the denaturation claim is a 3 pages short communication by Doty and
128
Rice published in 1955 [11]. With some emphasis, this paper can be said to
129
represent a sort of “parting of the Red Sea” as it marked the birth of a new
130
post-1955 paradigm on the preserved integrity of molecular information (after
131
DNA is brought to temperatures capable to irreversibly reduce its viscosity,
132
generally above 81°C, and consecutively cooled). Within a pre-1955 paradigm,
133
the DNA molecule appears to be divided into many smaller fragments upon
134
heating above 81°C while, within a post-1955 paradigm retrievable in part of
135
the literature, it undergoes only a marginal alteration which, at least according
136
to what has been stated by Doty in his 1955 paper, may also leave its molecular
137
weight unchanged or even determine strand disassociation. Quite remarka-
138
bly, heating above 81°C, under some fast cooling conditions requirements spec-
139
ified in a subsequent 1960 paper by Doty and coauthors [12], is deemed to be
140
even capable of bringing neat strands separation with halving of molecular
141
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weight. Given the discriminant role of the 1955 short communication, this pa-
142
per is carefully scrutinized in a subsequent section of the present study.
143
144
The aim of the present review is to analyze such apparent conflict be-
145
tween what has been reported in the more recent papers dealing with the de-
146
naturation claim and what has been found in previous seminal scientific pub-
147
lications of the decade 1950-1960, which have first investigated the matter of
148
thermal denaturation/degradation.
149
The overall focus of the present study is on answering the following main
150
research question:
151
based on the available scientific literature concerning the investigation of
152
thermal degradation of DNA, is it plausible to admit that an amplification of
153
genetic information, stored in the sequence of DNA, can be achieved by con-
154
secutive heating/cooling cycles with temperatures above 90°C?
155
To answer the question above a detailed scientific review is reported of
156
publications which have provided qualitative and quantitative evidence elu-
157
cidating if, how, and under which conditions, an onset of longitudinal se-
158
quence-breaking random fragmentation of DNA molecules is possible at the
159
PCR heating temperatures above 90°C. Based on the critical scientific review
160
and discussion of settling issues relevant to the plausibility of achieving am-
161
plification of DNA information by consecutive heating/cooling cycles at tem-
162
peratures above 90°C, this study is brought to a precise answer to the main
163
research question above and implications on PCR fidelity are drawn in a con-
164
clusive section.
165
Given the ever-increasing diffusion of PCR in forensic science and in kits
166
for diagnostic applications asseverated by the World Health Organization
167
(some of which commonly known as “PCR swabs), the purpose and signifi-
168
cance of the questions posed in the current study are promptly understood to
169
be of high relevance for public health policies as well as in forensic science.
170
171
172
1.1. Guide to reading subsections of this review
173
174
The present study is organized as follows. In Section 2 a scientific back-
175
ground is reported and discussed in three parts separately.
176
Subsection 2.1 presents a retrospective bibliographic survey
177
which starts from the account of the denaturation claim in Mullis’
178
1987 patent and parses all evidence about the denaturation claim
179
retrievable from the bibliographical references contained in the
180
main papers which have presented PCR.
181
Subsection 2.2 presents a first introductory review of three papers
182
of Doty et al. of the years 19551960 which have introduced
183
some controversial elements of interpretation of the effect of
184
DNA heating at high temperatures.
185
A third subsection, 2.3, reviews the pioneering studies from 1950
186
to 1960 on the effects of heating and of other physical and chemi-
187
cal agents, which mainly describe evidence against the
188
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denaturation claim and consequently raise questions on the fidel-
189
ity of PCR.
190
Focus of Section 2 is specifically kept over experiments capable to show
191
whether or not DNA complementary strands neatly separate as a consequence
192
of heating, and even more over the associated degree of preserved integrity
193
of molecular information upon repeated heating-cooling cycles with heating
194
above 80°C.
195
The organization of this review prosecutes as follows. Section 3 contains
196
a closer scrutinization and critical review of the papers by Doty and coworkers
197
in the years 1955-1960 and pinpoints key issues related to the denaturation
198
claim which are identified to be pivotal elements of controversy when com-
199
pared with contemporary results on DNA heating. In Section 4, such key is-
200
sues are discussed in the light of experimental evidence collected in this study
201
from the literature herein reviewed. Conclusions are finally drawn in Section
202
5.
203
204
205
206
207
2. Scientific background
208
2.1. The denaturation claim from PCR back to Doty and Rice (1955)
209
In Mullis and Faloona’s paper [4] few information is reported on the
210
DNA denaturation step. Among five methods therein presented, for methods
211
I and II the Authors state: The solution is brought to 100°C for 1 min, and is
212
cooled to 25 ° for 30 sec in a waterbath + 2 minand the same heating time and
213
temperature are used for method II. For methods III, IV, V and VI, heating is
214
reported at a temperature of 95° with time varying from 2 to 5 minutes. The
215
paper does not contain references which deal with the function of the DNA
216
denaturation associated with the specific methodology employed, except for
217
three footnote references [1315]. No differences of results are mentioned con-
218
cerning the use of the two different temperatures and times.
219
Since Mullis and coworkers in their first descriptions [3,4] do offer scarce
220
bibliographical support to the denaturation claim, a dedicated background is
221
reported in this section. Hereafter, excerpts from three references [1315]
222
mentioned in [3] and from those retrieved in Mullis’ patent [1617] are re-
223
ported, selected as far as they contain significant information concerning the
224
issue under scrutiny, i.e. relevant or relatable to the denaturation claim as
225
presented by K. Mullis. Following a philological rationale, these excerpts are
226
presented by publication date in reverse order:
227
In (Innis, 1988) [6], mentioned by Kary Mullis in 1990 [1] an an-
228
nealing reaction” is described comprising a step in which the mix-
229
ture was heated to 90°C for 3 min”. Also, brief accounts of PCR
230
thermal cycling and of denaturation at 93°C for 30 sec” are re-
231
ported as well as brief accounts of denaturing steps “at 80°C for 3
232
min” and “at 75°C for 5 min”.
233
In (Kwok et al., 1987) [15], where the authors deal with the appli-
234
cation of procedures related to PCR in search for identification of
235
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a virus deemed responsible for acquired immunodeficiency syn-
236
drome (AIDS), a denaturation step is mentioned, although no rel-
237
evant information is provided on the denaturation procedure
238
therein employed.
239
In (Scharf et al., 1986) [14] it is reported: 2 minutes of denaturation
240
at 95°C, 2 minutes of cooling at 37°C”.
241
In Saiki et al. (1985) [13] the information relevant to heating that
242
can be retrieved is the temperature of 95°C, and heating times of
243
2 minutes, 5 minutes or more, up to 10 minutes.
244
In (Gaubatz and Paddock, 1982) [16] a Heating and quick cool
245
step is reported in Fig. 5 therein which depicts an Hypothetical
246
approach for sequencing recombinant plasmid containing a cDNA. In
247
Section 4 therein it can be also read about heat denaturation and
248
quenching in ice”.
249
The 1979 paper by Caton and Robertson [17] reports: a novel tech-
250
nique is described for the production of pure, full-length influenza virus
251
ds DNA's corresponding to each segment of the influenza virus genome,
252
and suitable for molecular cloning and restriction endonucleaseand,
253
concerning heating, it is reported that Corresponding cDNA's
254
were […] boiled for 30 seconds and cooled on ice”.
255
The excerpts collected above constitute the almost entire body of textual
256
information on the heating/cooling step that is readily retrievable after a first
257
level bibliographic scan from [14]. For the sake of scientific reproducibility of
258
materials and methods related to the PCR denaturation step, this information
259
is not sufficient to allow prompt repetition of the same experimental methods
260
by other researchers; even more, this information is not even adequate to per-
261
mit a basic understanding of the elementary significance and purpose of tem-
262
perature change in a 20°C-wide range of critical values, i.e., from 80°C to
263
100°C in this step of the PCR scheme. Times of heating also frequently dif-
264
fer without explanation (from 1 minute to 1 hour or even more).
265
This substantial lack of information may let the reader presume that the
266
denaturation claim might have been considered established enough in the
267
specialized scientific community, at the time of these publication (i.e., in the
268
time frame 1979-1990), so as to not require additional descriptions or elucida-
269
tions.
270
A second level bibliographic investigation of references cited in the pa-
271
pers just mentioned [6, 13-17], proceeding from 1979 backwards, offers a few
272
additional elements about the denaturation claim.
273
Innis et al. [6] place PCR in the wake of refinements of DNA sequencing
274
techniques, which followed the methodology reported in a 1977 paper by
275
Sanger et al. [18]. Therein, concerning the denaturation claim, the only state-
276
ment retrievable is that a solution of DNA is heated to 100°C for 3 min”. This
277
procedure refers to the DNA sequencing method described in a paper by Air
278
et al. (1976) [19], which is actually based on heating at 100°C for 3 min and
279
which, in turn, is reported to correspond, with slight modifications, to a pre-
280
vious DNA sequencing method described by Sanger and Coulson (1975) [20].
281
In [20] the denaturation step is reported “to separate the newly-synthesized
282
strands from the template. The importance of denaturing is remarked and, con-
283
cerning heating, temperatures and time are indicated at 95°C as well as at 95
284
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to 100°C for 3 min. In presenting the principle of their synthesis method,
285
Sanger and Coulson cite Wu and Kaiser (1968) [21] who also refer in their
286
materials and methods section, when dealing with hydrolysis of DNA, of a
287
heating process of DNA mixtures to 100°C for 3 min”. Therein reference is
288
made to the enzymatic synthesis method of DNA proposed by Josse et al.
289
(1961) [22]. In this last paper, denaturation by heating is described with a
290
longer heating time: They specifically report Heating of calf thymus DNA for
291
30 minutes at 100° in a medium of low ionic strength results in collapse of the rigid
292
helical structure to a randomly coiled configuration.
293
It can be concluded from this first literature parsing that the practice of
294
heating steps along times from 1 to 5 minutes, reported by Mullis and Faloona
295
(1987) and by Innis et al (1988), can be indirectly traced back to heating prac-
296
tices already accepted in 1961 as documented by the paper of Josse et al. who
297
apply 100°C for the larger time of 30 minutes.
298
Josse et al. report that “The pattern of sequence frequencies was the same in
299
DNA synthesized with calf thymus DNA primer or with enzymatically prepared pri-
300
mer in which only traces of the native calf thymus DNA primer were present. The
301
pattern was also unaltered when the primer used was denatured by heating.
302
A fundamental point to be remarked concerning the paper by Josse et al.
303
(1961) is the notion of unaltered pattern of frequencies. In the first page of this
304
paper it is clearly explained, however, that pattern of frequencies refers to
305
the frequency of occurrence of nucleotides. Understanding that the notion of
306
unaltered pattern of frequencies of nucleotides is completely different from the
307
notion of unaltered pattern of nucleotides, a fundamental question to be formu-
308
lated for the sake of integrity of genetic information becomes the following:
309
To what extent the collapse entailed by such heating denaturation practices is re-
310
versible for the sake of the integrity of the pattern of the sequences of nucleotides?
311
312
Geiduschek and Holtzer in their 1962 review on light-scattering studies
313
for the characterization of DNA [24] also review the stability of DNA in aque-
314
ous solutions to acids and heat and summarize the findings achieved at their
315
time into the following sentences (see pages 489-490 therein): No distinction
316
between heat and acid denaturation is to be made. By suitable adjustment of these
317
variables, it is possible to deform DNA without change of molecular weight although
318
denaturation may under the proper circumstances be followed by degradation or even
319
aggregation”. Also, they write: The native helical structure of DNA can be dis-
320
rupted in a variety of ways not involving changes of its chemical constitution” and
321
specify that among other causes “addition of acid [..], heating […] are all capable
322
of producing denaturation”. A list of 13 references supports this last assertion
323
on heating denaturation. These references are examined according to their
324
ability to provide settling elements of evidence for answering to the key re-
325
search question of the present study: if, and how, at the PCR heating temper-
326
atures, above 90°C, an onset of longitudinal sequence-breaking random frag-
327
mentation of DNA molecules is possible, and under which conditions. The
328
works of this list of 13 references containing fundamental elements for an-
329
swering are reviewed in closer detail in Subsection 2.3 together with other
330
fundamental publications. Four of these 13 works are, instead, more briefly
331
reviewed hereafter (explaining the reason for such a briefer review).
332
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- The scientific content of the one-page conference proceeding by But-
333
ler, Shooter, and Pain containing their report at the 1955 Third Inter-
334
national Congress of Biochemistry in Bruxelles [25] is more broadly
335
presented and discussed in the six-pages publication by Shooter Pain
336
and Butler [9] on the journal Biochimica et Biophysica Acta submitted
337
just one month after the conference, on September 1955. This last sci-
338
entific publication is reviewed in detail in Subsections 2.3.6 and 3.1.
339
- From the 1955 book “The nucleic acids” edited by Davidson and
340
Chargaff, and contained among the 13 references by Geidusheck, no
341
specific conclusive information can be retrieved on the effect of heat-
342
ing, in terms of proof or disproof of the occurrence of sequence-break-
343
ing longitudinal fragmentation. In particular concerning heating at
344
100°C, in the fifth chapter on hydrolysis of nucleic acids by Loring
345
[26] on page 196 it can be read: The purine bases of DNA are easily
346
removed by mild acid treatment (heating the free nucleic acid in 2 % solution
347
at boiling water bath temperature for 10 minutes) apparently without com-
348
plete degradation of the original polynucleotide structure. The material re-
349
maining was early recognized as a complex substance. In Chargaff’s tenth
350
chapter of the same book [27], the distinction between denaturation
351
and degradation is discussed. Concerning the meaning of denatura-
352
tion, it can be read: “A mild, but persistent, mistreatment of a protein leads
353
to a state of malaise known, vaguely, as denaturation. The Author writes:
354
The line separating a denaturation product from a degradation product is
355
not clearly drawn; but one could define as denaturation products those sub-
356
stances whose preparation caused interference with the physical properties,
357
but not with the chemical composition, of the parent nucleic acid, while the
358
latter change will form part of the description of a degradation product.”.
359
This definition is understood to leave completely open the question
360
on whether the word denaturation contemplates or not a disruption or
361
a randomization of the genetic sequence by sequence-breaking fragmen-
362
tations and sequence-recombining aggregations. However, no specific re-
363
view or discussion of heating effects is reported in Chargaff’s chapter
364
and in the remainder of this book. Concerning molecular weight de-
365
cay as a primary indicator of possible longitudinal sequence-break-
366
ing fragmentation, the reassuring conclusion achieved by Reich-
367
mann, Bunce and Doty (1953) [28] is reported by Chargaff that the
368
adjustment of calf thymus DNA solutions (in 0.2 M NaCl), to pH 2.6
369
by dialysis did not affect the molecular weight (7,700,000), as determined
370
by light scattering.. This conclusion by Reichmann et al. quoted in ref-
371
erence [28] is closely reviewed in Subsection 3.2.2 together with the
372
underlying experimental evidence. Concerning stability to heat, even
373
in Jordan’s chapter [29], devoted to the physical properties of nucleic
374
acids, no decisive information is retrieved about the thermal stability
375
to longitudinal fragmentation of the DNA molecule, apart from a
376
mention of the protective effect of salt against heat denaturation, as
377
found in Thomaswork [30-31]. It can be read in this chapter: A
378
further protective effect of salts against heat denaturation of DNA solutions
379
has been observed by Thomas. For calf thymus DNA in sodium chloride so-
380
lutions of various concentrations, some denaturation occurs at room
381
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temperature at <10-4 M and below 70° in 10-2 M. Denaturation is strongly
382
inhibited even at 100° C. in 10-1 M and M solutions. It is claimed that heat
383
denaturation occurs stepwise and that the critical temperature for each step
384
varies for DNA from different sources. Thus DNA from starfish testis in 10-
385
2 M sodium chloride undergoes the first step in denaturation at 55° C. com-
386
pared with 70° C. for that from calf thymus.. In particular, the statement
387
that “Denaturation is strongly inhibited even at 100° C. in 10-1 M and M
388
solutions” is a quotation of Thomas (1954) [29,31]. Concerning this last
389
statement by Thomas, it should be remarked that Thomas detects de-
390
naturation by changes in optical density at 260 m and that this meas-
391
ure in not conclusive neither in proof nor in disproof of occurrence of
392
sequence-breaking longitudinal fragmentation of the DNA molecule
393
at 100°C in presence of these salt concentrations.
394
- Finally, the two papers by Zamenhof and coauthors [32,33], among
395
the 13 cited by Geiduschek and Holtzer concerning heating degrada-
396
tion and denaturation, are also not reviewed in detail since these two
397
works just extend measures of viscosity drop and bacterial transfor-
398
mation inactivation reported by Zamenhof et al. (1953) [8] and do not
399
provide conclusive evidence in proof or disproof of the possibility of
400
the onset of disruptive longitudinal sequence-breaking random frag-
401
mentation of DNA molecules in addition to the fundamental ele-
402
ments of evidence already reported by Zamenhof in [8]. For this rea-
403
son, in Subsection 2.3.3 only the scientific publication by Zamenhof
404
et al. (1953) is reviewed.
405
- The remaining 9 references quoted by Geiduschek and Holtzer are all
406
reviewed in closer detail in the next subsections.
407
408
2.2. The pivotal papers of Doty et al. of the years 19551960
409
Continuing the literature review by keeping a reverse chronological or-
410
der and a focus on possible evidences of molecular integrity of the sequences
411
of nucleotides in a same strand at high temperature and/or of possible
412
strands separation, the bibliographic scan runs across three papers by Doty
413
and coworkers published between 1955 and 1960 [11,10,23], and already men-
414
tioned in the introduction. These papers report results of physical and chem-
415
ical analyses specifically devised to follow the molecular weight changes of
416
DNA during and after a heating/cooling cycle in order to attempt to under-
417
stand how depolymerization and possible strand separation progress as a
418
consequence of heating. The conclusions of these three papers by Doty re-
419
garding the consequences of DNA heating at 100°C deserve to be remarked
420
since they entail significant elements of mutual disagreement (i.e., conflict
421
among conclusions stated in these same three papers) and of conflict with the
422
conclusions of studies on DNA heating published before 1955. Some of these
423
elements of disagreement are reported hereafter.
424
In particular, Doty et al. (1960) [12] conclude their study reporting, in a
425
final summary, a very general statement: “When solutions of bacterial DNA are
426
denatured by heating and then cooled, two different molecular states can be obtained
427
in essentially pure form depending on the choice of conditions, that is, rate of cooling,
428
DNA concentration, and ionic strength”. They continue: “One state corresponding
429
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to fast cooling consists of single stranded DNA having about half the molecular
430
weight of the original DNA. The other state corresponding to slow cooling consists
431
of recombined strands united by complementary base pairing over most of their
432
length. This form has as much as 50 per cent of its original transforming activity and
433
is called renatured. The statement above is remarkably very general since,
434
without specifying the heating temperature and the heating time, it depicts a
435
single stranded product resulting from complete disassociation of all hydro-
436
gen bonds between complementary strands into molecular weight halving.
437
We will refer to this picture as the high temperature heating + fast cooling disso-
438
ciation claim. A second point of remark for the 1960 statement above is that it
439
turns out to be in open conflict with the conclusions by Doty and Rice in two
440
papers, one published in 1957 [23] and the other one published as a prelimi-
441
nary note in 1955 [11]. Their preliminary 1955 conclusion is that, upon heating
442
a neutral saline DNA solution to 100°C for 15 minutes, the product is found
443
to have the same molecular weight as the native DNA [11]. Such conclusion is
444
further specified in 1957 to be inferred from light scattering studies, as the
445
Authors write that, upon observing the effects of heating of four calf thymus
446
DNA preparations along exposure times of one hour and more at tempera-
447
tures from 89°C up to 100°C, “the molecular weight remains unchanged according
448
to light scattering studies [11]. We will refer to the paradigm introduced in
449
these last 1955 and 1957 papers by Doty and Rice as the 100°C stable molecular
450
weight claim. Although the Authors include in [11] bibliographical references
451
to previous studies, such as the study by Goldstein and Stern [7] and Zamen-
452
hof et al. [8], which bring instead to the opposite conclusion of DNA mole-
453
cules thermally fragmenting in aqueous solutions already at temperatures not
454
higher than 81°C, the 100°C stable molecular weight claim is remarkable since
455
they make no mention of the existence of such a macroscopic conflict with the
456
previous literature. Even more remarkably, except for the 1955 preliminary
457
note, no subsequent mention is made by Doty et al. in [10] and [23] of Gold-
458
stein and Stern’s study among the referenced works, so that the trace of their
459
fundamental contribution is lost.
460
Additional elements of perplexity rise from reading in Doty et al. (1960)
461
[12] statements that appear to be in open conflict with both the high tempera-
462
ture heating + fast cooling dissociation claim and the 100°C stable molecular weight
463
claim. For instance, the Authors report in [12] that heating pneumococcal
464
DNA of molecular weight 8.2 million at 100° for 10 minutes in standard saline
465
citrate and subsequently quickly cooling this material, they find a molecular
466
weight of 2.0 million (see page 471 therein). Even excluding, simplistically,
467
any considerations of polydispersity, this evidence is neither compatible with
468
the first claim (which would require a decrease of molecular weight by a fac-
469
tor of one half) nor with the second claim.
470
471
The literature showing evidence of thermally induced DNA fragmenta-
472
tion and the characteristic temperatures above which this phenomenon is ob-
473
served is examined in the next subsection 2.3. The discrepancies determined
474
by the 1955 and 1957 papers by Doty and Rice with previous findings are
475
specifically examined and critically reviewed in the subsequent Section 3 of
476
the present study.
477
478
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2.3. Pioneering pre-1961 studies on DNA heating not authored by Doty
479
Prosecuting the literature scan before 1955, results are found implying
480
significant elements of divergence related to the high temperature heating + fast
481
cooling dissociation claim and to the 100°C stable molecular weight claim. This
482
group of references are presented hereafter in progressive chronological
483
order.
484
485
486
2.3.1. Evidence of viscosity drop and of heat turning fibrous material into flocculent
487
precipitate
488
Strong and consistent evidence of DNA thermal degradation resulting in
489
the fragmentation into much smaller molecular products (thus incompatible
490
with a hypothesis of orderly disassociation or with one of unchanged molec-
491
ular weight) had clearly emerged in several papers between 1950 and 1960.
492
Milestone results of experiments on thermal depolymerization showing this
493
fragmentation evidence are described by Goldstein and Stern in 1950 [7]. The
494
Authors report the following settling experimental observation Preliminary
495
tests showed that a highly viscous aqueous solution of sodium desoxyribosenucleinate
496
loses its viscosity when it is heated almost to the boiling point and then permitted to
497
cool to room temperature. Upon adding the solution to 1.5 volumes of ethyl alcohol a
498
flocculent precipitate is formed instead of the fibrous material yielded by "native" nu-
499
cleic acid preparations. Concerning viscosity, Fig. 12 therein (reproduced in
500
Fig. 11 of Section 4.1) shows that a 0.1% solution of purified calf thymus DNA
501
in a barbital buffer at pH 7.2, heated just one minute at 100°C, experiments an
502
irreversible drop of relative viscosity, from a value of 3.4 to a value below 1.4
503
(see Fig.7TEMP in Section 4.1). Besides, the same figure shows a similar drop
504
to a value, close to 1.4, is obtained by a 2 minutes heating at 90°C.
505
Notably, Goldstein and Stern warn that an experiment that fails in detect-
506
ing evidences of depolymerization (produced either sonically or thermally) is
507
ultraviolet absorption spectrophotometry. Specifically, concerning changes
508
upon heating in ultraviolet spectrum absorption of solutions of tetrasodium
509
salt of DNA (therein indicated by the acronym STN), they report: “The exami-
510
nation in the Beckman spectrophotometer of a 0.048% STN solution in distilled water
511
(after dilution to 0.008%) which had been heated for 15 minutes at 100° showed no
512
change in the position of the maximum at 259 m
or of the minimum at 231 m
,
513
although the relative viscosity had fallen from 3.11 to 1.35 as a result of this treatment.
514
The rise in optical density from 1.28 to 1.46 at 259 m
which was observed in this
515
experiment may possibly be due to the fact that it was performed in the absence of a
516
buffer system.
517
Goldstein and Stern also warn (see section therein Effect on Absorption
518
Spectrum”) that when the optical density absorption spectrum is observed in
519
the wave lengths range from 225 to 300 m, changes are detected only if the
520
solvent is distilled water (“unbufferedsolution) while in the buffered solu-
521
tions no changes are observed. Such findings find confirmation in a study by
522
Blout and Asadourian (1954) [35] showing that the intensity of ultraviolet ab-
523
sorption of the sodium salt of DNA is dependent on the ionic strength. The
524
intensity of absorption is highly pH dependent, in the absence of salt, in the
525
pH range 3 to 12. These and other results therein reported concerning various
526
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heating tests between 60 and 100°C lead the Authors to the conclusive obser-
527
vation: ‘‘The depolymerization of desoxyribosenucleic acid by thermal treatment is of
528
special interest in view of the high temperature coefficient and, hence, large energy of
529
activation, observed in the range between 60 and 100°C (see Fig. 12). While this phe-
530
nomenon is similar to the heat inactivation and denaturation of proteins (cf. ref. 22),
531
there exist important differences. Thus, the nucleic acid molecules appear to be split
532
into many small fragments on heating while the thermal denaturation of proteins, as
533
a rule, does not lead to a significant alteration of their molecular weight, although in
534
some instances aggregation will occur under these conditions (cf. ref. 22). This would
535
seem to indicate that the bond strength of the sugar-phosphate links in the polynucle-
536
otide chain is smaller than that of the peptide bonds in proteins.’’
537
538
In the same year 1950, Miyaji and Price [34] find confirmation of the vis-
539
cosity drop induced by heating. They also study the effect of NaCl addition
540
coming to the following conclusion: “The viscosity of aqueous solutions of sodium
541
thymonucleate is reduced both by heating and by addition of sodium chloride and other
542
salts. If, however, the nucleate solution is heated in the presence of a sufficiently high
543
concentration of salt, there is no further decrease in viscosity beyond that induced by
544
the salt. The protective effect of the salt is reversible, for if the salt is removed by dial-
545
ysis from the heated nucleate-salt mixture, the residual aqueous solution of nucleate
546
again suffers a marked reduction in viscosity on heating”. They also find the singu-
547
lar phenomenon above 85°C that “At temperatures over 85°C, mixtures of nucle-
548
ate and salt sometimes yielded a small amount of white precipitate.”.
549
Miyaji and Price highlight the importance of discriminating whether the
550
drop in viscosity is due to 1) either fragmentation of the DNA molecule or 2)
551
to coiling of the DNA molecule with reduction of molecular radius yet in ab-
552
sence of fragmentation, or even to a combination of the two phenomena.
553
554
555
2.3.2. Evidence of thixotropy induced by heating
556
557
Also in 1950, Zamenhof and Chargaff [36] find that heating a 0.3 per cent
558
solution of DNA at 86°C for 90 minutes makes the specific viscosity,  ,drop
559
from 30.3 to 3.9. They find that the solution degraded by heating can repoly-
560
merize but show that a DNA preparation degraded by heat (similar to the
561
degradation produced by other agents such as acid and alkali) acquires a
562
marked thixotropy (i.e., reduction of viscosity determined by forced flow and
563
viscosity regain as the forced flow is arrested). They also show, conversely,
564
that an intact DNA preparation exhibits no thixotropy since its viscosity re-
565
mains unaltered upon subjecting it to forced flow. They observe that this effect
566
of induced thixotropy is irreversible since it cannot be removed by repolymer-
567
ization. They conclude that their experiments show that “original preparations
568
of desoxypentose nucleic acids possess unique physical properties (non-thixotropic
569
viscosity, regular temperature-viscosity relationship) which, once lost, cannot be re-
570
gained by repolymerization”. They specify that such phenomena of irreversible
571
acquisition of thixotropy “were observed with desoxypentose nucleic acid prepara-
572
tions from calf thymus and from yeast, depolymerized not only by acid or alkali but
573
also by heat”. Also, with Goldstein and Stern they are among the first who ex-
574
tend the concept of irreversible denaturation from the field of proteins to that of
575
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another group of macromolecules, the nucleic acidsand likewise remark the irre-
576
versible character of the transformations produced in DNA by heating.
577
578
579
580
581
582
2.3.3. Evidence of vanishing of transforming activity paired by viscosity drop
583
584
A biological confirmation of the simultaneous onset, at heating tempera-
585
tures higher than  = 81°C, of irreversible viscosity drop phenomena paired
586
by biological inactivation phenomena is reported by Zamenhof, Alexander
587
and Leidy in 1953 [8]. The Authors extract and purify DNA from a culture of
588
Hemophilus influenzae that has resistance to streptomycin. They add dilu-
589
tions of these aqueous solutions of purified DNA from streptomycin-resistant
590
colonies to bacterial suspensions obtained from cultures derived from strains
591
characterized by initial absence of resistance to streptomycin, under controlled
592
37°C temperature and time. Subsequently, they add streptomycin (SM) and
593
detect, by observing the quantity of surviving bacteria, the degree of re-
594
sistance to streptomycin induced in the non SM-resistant colonies by DNA
595
addition. Thanks to dilutions they can also measure the degree of biological
596
activity of the extracted DNA solutions.
597
As well known, biology interprets this phenomenon of induced resistance
598
as a parasexual phenomenon, known as Griffith bacterial transformation ac-
599
tivity, thanks to which the bacterium that initially does not have resistance to
600
streptomycin, upon entering in contact with the purified DNA solution «ac-
601
quires» the genetic code contained in the DNA molecules thus receiving the
602
necessary instructions to build the polysaccharide bacterial capsule that gives
603
the increase in resistance to streptomycin, and transmits it to its own lineage.
604
They perform part of their experiments with H. Influenzae after subjecting the
605
DNA extract to heating at a variable T temperature in a pH 7.4 buffer with
606
0.14 M NaC1 and 0.015 M sodium citrate (upon reporting the temperature to
607
23°C before each activity assay). They find that viscosity and the activity are
608
practically unaffected by 1 hour heating to temperatures (T) as high as 76-81°C
609
while, in a temperature range (T) between 81 and 90°C heating, induces a
610
more than significant reduction and, by further increasing (T), they see a drop
611
to practically zero of the phenomenon of bacterial transformation activity.
612
Points on the graph of Fig. 1 by the Authors on page 379 [8], concerning
613
measures of relative bacterial transformative activity (in percentage), have
614
been digitized; the resulting numerical values, converted from logarithmic
615
scale, are reported in Table 1 herein. The values of Table 1 are plotted in Fig.
616
1 reporting, on the x horizontal axis, the heating temperature of DNA in °C
617
and, on the ordinates, the percentage of residual bacterial transformation ac-
618
tivity employing a linear scale in place of the logarithmic scale of the original
619
figure.
620
621
622
623
624
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625
Table 1. Numerical values obtained by digitalization of points in Fig. 1 in [8]
626
concerning the stability of the transforming principle preparation to heat.
627
Heating temperature
Activity
[°C]
[%]
24.92
100.000
38.84
100.000
60.85
100.000
76.05
100.000
80.91
100.000
81.39
14.908
84.84
4.040
91.12
0.392
91.68
0.146
89.06
0.100
99.38
0.001
99.35
0.001
1 Values of activity have been converted from the logarithmic scale of the
628
original figure.
629
630
631
Figure 2. Activity vs. heating temperature reproduced from Fig. 1 in [8]. Or-
632
dinates are represented by an ordinary linear scale in place of the logarith-
633
mic scale of the original figure.
634
The evidence found in [8], clearly represented by Fig. 2 herein, is that,
635
upon subjecting a purified DNA aqueous solution to a preliminary heat-
636
ing/cooling cycle bringing it to a variable temperature (T) and back again to
637
23°C, the residual bacterial transformation activity is stable when T ranges be-
638
tween 25 and 80.9°C. When the heating temperature T reaches 81.4°C, the
639
transforming activity is reduced to 15%, and, by increasing the heating tem-
640
perature, the activity continues to decay rapidly to zero, so that 90°C it is ba-
641
sically absent.
642
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A further fundamental result in [8] concerns stability of DNA viscosity
643
after heating. Upon measuring viscosity in the same pH 7.4 buffer at 23°C,
644
they find that the viscosity and the activity are practically unaffected by 1 hour
645
heating to temperatures as high as 76-81°C.”.
646
Concerning the possibility that heating or acidity may just induce molec-
647
ular contraction and not depolymerization Zamenhof et al. also write: It has
648
recently been suggested (22, 26, 27) that the decrease in viscosity of DNA solution
649
upon mild H+ treatment is due to the change in asymmetry caused by the contracting
650
of the molecule rather than by actual depolymerization. This may also be true for the
651
mild heating. The contraction may be made possible by the breakage of labile bonds
652
(such as hydrogen bonds) under the action of thermal oscillations (40, 41). At higher
653
temperatures, actual depolymerization may occur (32).
654
655
656
2.3.4. Evidence of depolimerization by ultraviolet light measurements
657
658
Thomas in his 1954 publication [31] on the study of the ultraviolet absorp-
659
tion spectrum of neutral saline NaCl (unbuffered) solution of desoxyribonu-
660
cleic acids (DNA) isolated from calf thymus, starfish (Asterias Glacialis) testi-
661
cles and red frog (Rana Temporaria) testicle devotes an entire section, titled “Ef-
662
fect irreversibles de la temperature sur le spectre U.V”, on the effects of heat-
663
ing on changes of optical density measures. Besides finding that this measure
664
is strongly sensitive to the NaCl concentration, he plots the graphs of the func-
665
tion relating the absorption at 260 m
versus the preheating temperature and
666
he finds sudden increases of the optical density at temperatures ranging be-
667
tween 55°C and 80°C with intermediate plateau regions in which the absorp-
668
tion remains constant. For calf thymus DNA he finds a maximum upper limit
669
for stability against depolymerization corresponding to a temperature around
670
80°C, in agreement with Goldstein and Stern (although these authors are not
671
quoted by Thomas).
672
It is worth observing that the detection by Thomas of changes in optical
673
density as function of heating temperature are also in agreement with the ex-
674
planation by Goldstein and Stern since Thomas employs unbuffered solu-
675
tions.
676
677
678
2.3.5. Detection of depolymerization by staining
679
680
Measurements indicating depolymerization of DNA available in 1954 are
681
summarized by Kurnick [39] and recalled in the following list:
682
- reduction in viscosity;
683
- reduction in rate of sedimentation in the ultracentrifuge;
684
- increase in ultraviolet absorption (detectable in unbuffered solu-
685
tions);
686
- lowering of PH;
687
- formation of acid-soluble and dialyzable products.
688
Based on his previous study [40] where he could show that methyl green
689
selectively stains only highly polymerized desoxyribonucleic acid, and fails
690
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to stain, to any significant extent, depolymerized desoxyribonucleic acid and
691
ribonucleic acid, Kurnick introduces a new methodology for studying depol-
692
ymerization induced by heat [39]. From experiments on calf thymus DNA,
693
among which ultracentrifugation patterns of DNA solutions, with and with-
694
out 0.02% methyl green, heated to 80° for 7 hours, and to 100° for 1 hour or 2
695
hours as well as on unheated DNA, he finds that Heat and enzymatic depoly-
696
merization of DNA produce certain effects in common: both reduce the viscosity of the
697
solution rapidly […], increase the ultraviolet absorption […] and reduce methyl green
698
affinity”. He remarks that “These changes are maximal before any dialyzable, acid-
699
soluble oligonucleotides are formed […] and nearly maximal before reduction in sedi-
700
mentation rate appears. He adds: The products of heat and initial enzymatic di-
701
gestion (and of sonic depolymerization) are large molecules which, as judged by their
702
sedimentation velocity, are probably still quite asymmetric rods. This hypothesis is in
703
better accord with the unaltered sedimentation rate (despite marked reduction in vis-
704
cosity) than the alternative suggestion [citation of Creeth Gulland and Jordan (1947)
705
[41] and of Conway and Butler] that heat has produced collapsed molecules of the
706
original molecular weights. Such collapsed molecules would show a great reduction
707
in viscosity, but would also show considerable change in sedimentability.”
708
He conclusively provides an explanation of the heating degradation at
709
100°C in terms of “bombardment by the solvent molecules”: Thus, at l00°C, a mol-
710
ecule with very low intrinsic viscosity, but of a considerable size as characterized by
711
its non-dialyzability, sedimentability and affinity for methyl green […] is stable. This
712
suggests that the increased bombardment by the solvent molecules at elevated temper-
713
ature snaps the rigid molecule into still large units, but of such lesser size as to be
714
stable when subjected to the bombardment characteristic of this temperature.
715
716
717
From the background above on the studies on thermal depolymerization
718
published between 1950 and 1954, it consistently emerges that heating at tem-
719
peratures above 81°, or just above 81°, ordinarily produces depolymerization
720
(or disaggregation) in purified DNA solutions. For completeness, it is worth
721
adding that a residual possibly conceivable alternative justification explaining
722
drop in viscosity could be the hypothesis considered by Creeth Gulland e Jor-
723
dan (1947) [41] of a coiling of the DNA molecule capable of “reducing the mo-
724
lecular asymmetry but not the molecular weight”. It should be pointed out, how-
725
ever, that such a coiling hypothesis is drawn in [38] to explain decrease in vis-
726
cosity observed after addition of NaCl, occurring without production of titrat-
727
able groups and that in [41] no heating experiments are carried out.
728
729
730
2.3.6. Estimates of molecular weight from viscosity-sedimentation experiments
731
732
An important contribution comprehensively accounting for most of the
733
previously published literature on the effect of DNA heating, confirming
734
Goldstein and Stern’s conclusion on DNA fragmentation above 81°, is a phys-
735
ical chemical study by Dekker and Schachman (1954) [42]. This study, while
736
essentially focused on the assessment of different investigated models for the
737
possible macromolecular structure of DNA (among which also models differ-
738
ing from the Watson and Crick double helical chain [43]), is important for the
739
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sake of understanding the effect of heating, since it reports a simple quantita-
740
tive estimate of the molecular weight change before and after heating due to
741
fragmentation, by a calculation from sedimentation and viscosity data before
742
and after heating.
743
Dekker and Schachman do not exclude ab initio a possible coiling of the
744
DNA molecule determined by heating and they are the first to point out that
745
paired measurements of viscosity and sedimentation rate of dilute solutions
746
of DNA and heated DNA could differentiate between a disorganization of the
747
DNA molecule without a change in molecular weight (coiling), on one side,
748
and a process in which there is a degradation into much smaller pieces, on the
749
other side. Their key reasoning is that if, upon heating, the DNA molecule is
750
divided into smaller fragments, the viscosity must decrease and the sedimen-
751
tation coefficient must decrease as well, while, in case heating should produce
752
just a coiling of the molecule, unlike viscosity the sedimentation must instead
753
increase.
754
Based on their experimental measurements, the Authors make the funda-
755
mental observation which compels them to exclude a hypothesis of thermal
756
coiling as they write: The reduced viscosity of a 0.005 per cent solution heated at
757
pH 7 for 15 minutes at 100°C. decreased from 30 (gm/100 cc)-1 to less than 1.0
758
(gm/100 cc) -1. Instead of an increase in the sedimentation rate, which would be ex-
759
pected from the amount of coiling necessary to produce such a large drop in viscosity,
760
we found that measurements at 0.005 per cent DNA showed that the heated material
761
had a sedimentation coefficient of about 6S, whereas the unheated preparation had a
762
value of nearly 20S. These experiments provide proof that the molecular weight of
763
DNA changes from about 5106 to 5104 as a result of this mild heating procedure.
764
In the previous quotation, uppercase S indicates the Svedberg unit which cor-
765
responds to  where stands here for seconds. It is also worth re-
766
calling that the reduced viscosity, , is related to the viscosity through the
767
following relations:  
where is the concentration and  is the
768
specific viscosity  , being  the relative viscosity defined
769
as the ratio of solution viscosity to solvent viscosity . The intrinsic viscos-
770
ity  is obtained by extrapolation of reduced viscosity against concentration
771
at infinite dilution, , viz.: 

. [5,44].
772
773
The proportion behind this simple quantitative estimate by Dekker and
774
Schachman can be presented as follows. Denoting by the average weight of
775
the DNA molecule, or of its fragment produced upon heating (assuming here
776
introductively and simplistically that all molecules or fragments have the
777
same weight and molecular hydrodynamic radius ) the following direct pro-
778
portionality holds
779
780
(1)
781
where is now the sedimentation constant, the viscosity and is the symbol
782
of direct proportionality. References for the proportion above, which is
783
obtained combining Svedberg’s equation [45] with Stokes-Einstein’s equation,
784
can be found in treatises of physical chemistry of macromolecules such as
785
Barrow’s treatise (see, e.g. [46], Chapter 20 and, in particular, page 659, Eq. (23)).
786
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Fundamental more advanced investigations, contemporary to those years,
787
based on careful consideration of the hydrodynamic Stokes’s law, empyrical
788
data for long chain polymers, dimensionless analysis and investigation of
789
universal constant for flexible long chain polymers such as DNA can be found
790
in (Mandelkern et al., 1952) [47] and related references.
791
Dekker and Schachman’s estimate stems from the simplistic consideration
792
that the hydrodynamic radius remains unchanged upon heating so that, denot-
793
ing by subscripts and the quantities relevant to native DNA and to DNA
794
possibly fragmented by heating, respectively, one infers from Eq. (1) setting
795
:
796
797
798



(2)
799
800
801
Shooter, Pain and Butler (1956) [9] also dedicate a study to examining the
802
effect of thermal degradation above 80°C on DNA. Based on their experiments
803
they propose some simple graphical models suggesting the effects of heating
804
on DNA (see the reproduction in current Fig.3).
805
806
807
Figure 3. Handmade reproduction of Fig.4 by Shooter et al. (1956) [9] ‘Sug-
808
gested effect of heating’
809
810
They conclude their study stating that after 15 minutes heating at 100°C,
811
DNA samples always produces thermal degradation. They remark that the
812
extent of degradation and the distribution of the size and shape of the frag-
813
ments produced depends on how DNA is prepared.
814
815
816
817
818
819
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2.3.7. Evidence of irreversibility of heating degradation from titration analyses
820
821
Titration analyses although do not provide direct quantification of mo-
822
lecular weight decrease and direct evidence of possible onset of disruptive
823
longitudinal sequence-breaking random fragmentation of DNA molecules,
824
can detect however some irreversible features occurring during the heating
825
process and the quantity of broken hydrogen bonds and for these reasons
826
part of these works on titration relevant to DNA heating are hereafter re-
827
viewed.
828
The study by Cavalieri and Rosenberg (1957) [48] reports of potentiom-
829
etric titration analyses on DNA solutions using calomel and glass electrodes.
830
The study ascertains the conditions under which hydrogen bonds in DNA
831
can be cleaved reversibly by heat, and the conditions under which heat bonds
832
cleave spontaneously and irreversibly. Concerning the temperature at which
833
irreversible phenomena are observed the Authors report: A temperature is
834
finally reached (about 70° in 0.017 M NaCl, for example) at which denaturation oc-
835
curs without titrating any bases, and cannot be prevented by heating in buffers of
836
any pH. In such cases, the original H-bonds of DNA must all be thermally and irre-
837
versibly cleaved.”.
838
Cox and Peacocke (1956) [49] prior to presenting their results on electro-
839
metric titration well summarize the contemporary debate on the stability of
840
the molecular weight to heating: When sodium deoxyribonucleate is heated in
841
neutral aqueous solution, irreversible changes occur above a critical temperature
842
which varies with the source of the deoxyribonucleate and its method of extraction.
843
These irreversible changes, which often take place over a temperature range of only a
844
few degrees, include: a drop in the viscosity; an increase in the ultraviolet absorption;
845
a displacement of the spectrophotometric titration curves; the appearance of new in-
846
frared absorption bands; changes in sedimentation constant; and displacement of the
847
titration curves. Some investigators deduced a decrease in molecular weight from
848
sedimentation and viscosity measurements after the nucleate had been heated in wa-
849
ter [(Dekker and Schachman, 1954) [42, (Sadron, 1955) [51], and in salt [(Shooter et
850
al., 1956) [9]], whereas others reported [(Doty and Rice, 1955) [11], (Sadron, 1955)
851
[51] no change in molecular weight after heating in the presence of sufficient sodium
852
chloride. (notice that, in the quotation above, text references originally re-
853
ported in superscript format have been converted into the current squared
854
brackets format adding also author and year). They summarize in Fig.2
855
therein that the percentage of ruptured hydrogen bonds in herring-sperm in
856
a (0.15%, 0.05 M -NaCl) solution heated along 1 hour at 95°C is 100%. More-
857
over, on the basis these titration experiments the Authors infer evidence for
858
the random nature of the heat-denaturation process”.
859
We incidentally remark that the statement by Cox and Peacocke (1956)
860
[49] according to which (Sadron, 1955) [51] would report no change in molec-
861
ular weight after heating in the presence of sufficient sodium chloride, ap-
862
pears to be incorrect as the data in this publication by Sadron show in all cases
863
a decrease of the molecular weight after heating except for just one case where
864
it is reported even an increment of molecular weight. This issue is examined
865
in detail in Subsection 2.3.9 dedicated to the light scattering measurements by
866
Sadron on heated DNA.
867
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In 1957 Cox and Peacocke [52] specify the limit of titration in detecting
868
changes in molecular weight: ionizing radiations invariably and heat, under cer-
869
tain conditions also cause changes in molecular weight but this degradative aspect of
870
their action will not be considered further here, if only for the reason that the titration
871
curves can only just detect a release of one secondary phosphoryl end group in 50
872
nucleotides.
873
Most importantly, Cox, Overend, Peacocke and Wilson in their publica-
874
tion on Nature (1955) [5], reaffirm it is only changes in the intrinsic viscosity
875
which are of significance in the estimation of molecular size and shapeand propose
876
the employment of the following expression, usual for high weight polymers,
877
relating intrinsic viscosity to the is the viscosity-average molecular
878
weight,
879

(3)
880
where and are constants for homologous polymers. Based on their ex-
881
perimental data the Authors compute . Remarkably, agreement with
882
this value is found by the same Doty and coworkers who later compute in
883
1960 [12] from two DNA preparations from Diplococcus Pneumoniae DNA
884
and Escherichia Coli two determinations of and whose average value is
885
 and . The exponent close to unity signifies that the relation
886
between viscosity-average molecular weight and intrinsic viscosity is almost
887
linear.
888
Use of this formula with the intrinsic viscosity data, measured before and af-
889
ter a heating treatment, allows to compute the drops from the molecular
890
weight of the unheated molecule to the weight of the molecule after heat-
891
ing, , by the following formula:
892



(4*)
893
On the basis of light scattering experiments and viscosity measurements on
894
DNA degraded by gamma rays, Peacocke and Preston (1958) [50] improve
895
the precision of the determination of coefficients and in (3) they find a
896
round value  for the exponent and find  They find that the
897
weight-average molecular weight is related to intrinsic viscosity by:
898

(5)
899
Most importantly, they find a remarkable consistency of the value
900
obtained from (5) with countercheck values obtained by light scattering
901
measures, so that (5) can be considered a reliable choice for determining mo-
902
lecular weight from intrinsic viscosity, substitutive of light scattering meas-
903
urements.
904
905
Employing the data reported by Shooter et al. (1956) [9] concerning meas-
906
urements before and after 15 minutes heating at 100°C on different samples
907
and also in absence of salt (see Table I therein), the viscosity-based average
908
weight of the heated DNA molecules is computed from (4) to range between
909
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1.45% and 5.01% of the average weight before heating, what signifies a depol-
910
ymerization in considerably shorter fragments.
911
912
913
2.3.8. Models for random degradation matching experimental data
914
915
Kinetic molecular models provide a valuable mean for computing the
916
number of scissions (fragmentations in shorter segments) that the DNA mol-
917
ecule undergoes during a heating experiment and the rate of fragmentations,
918
i.e. the number of fragmentations per unit of time, based on diagrams of mo-
919
lecular weight plotted vs. time.
920
Peacocke and Preston (1958) [50], proceeding from viscosity and light
921
scattering experiments on DNA samples degraded after exposure at different
922
doses of gamma rays, find that internucleotide phosphodiester bonds in DNA
923
are ruptured by a random fragmentation process. Fragmentation is the pro-
924
cess which divides the molecule into shorter segments of lower molecular
925
weight. They experimentally find for DNA an almost quadratic relation (ex-
926
ponent 1.85) between the radiation dose and the intrinsic viscosity  in-
927
stead of the linear relation characteristic of single strand polymers. From such
928
fundamental difference they infer that in DNA (and in any other similar dou-
929
ble stranded polymer) the fragmentation of the molecule in a fragmentation
930
process must be generated by a double breakage mechanism. This mechanism
931
consists of the breakage of two intra-chain longitudinal bonds each located at
932
two facing nucleotides situated at the same longitudinal position of the mole-
933
cule but belonging to opposite strands. This mechanism is mathematized in
934
the context molecular weight distributions [53]. In this context distinction is
935
made between the number-average molecular weight which is the arith-
936
metic mean of the weights, and the weight-average molecular weight
937
which is the weighted arithmetic mean in which the molecular weights them-
938
selves as the averaging weights. For a uniform distribution while for
939
a random distribution (i.e., a distribution generated from random fracture of
940
an infinite chain) .
941
The considerations of Peacocke and Preston are conveniently summa-
942
rized hereafter in the format of proportionality relations. The weight-average
943
molecular weight and number-average molecular weight are related to
944
the number of fragmentations per original number-average molecule:
945
(6)
946
where the 0 subscript indicates the original molecular weight of the molecule
947
before fragmentation starts. We recall that in special cases For a higher num-
948
ber of molecules and fractures ( relation above can be represented as
949
the proportionality relation:
950
(7)
951
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For degradation by radiation of single strand polymer chains it is ordi-
952
narily expected that the probability of single chain break and the radiation
953
dose are related to fragmentation by:
954
(8)
955
what should imply from (7) that
. Peacocke and coworkers find instead,
956
experimentally, that after a transitory range of exposition the proportionality
957
holds:
. They explain in terms of probability distributions such evi-
958
dence, unusual for single stranded polymers, to be the consequence of a frag-
959
mentation process in which a fragmentation is produced when two intra-
960
chain scissions at opposite strands in two facing nucleotides occur (or in two
961
proximal nucleotides). The probability of this double-step process is shown to
962
follow the proportionality , so that, owing to the previous proportion-
963
ality relations, one has:
964
(9)
965
Owing to the previous equation and to (7) one finally has for DNA and
966
for any double stranded polymer the theoretical prediction:
967
968
or equivalently
(10)
969
It is important to highlight the difference between the previous relation
970
(10) and the corresponding relation for single-stranded polymers which reads
971
instead:
972
973
(11)
974
Peacocke and Preston find that for gamma-rays degradation of DNA re-
975
lation (10) is closely respected and consider this as evidence that the fragments
976
ensuing from gamma-ray degradations are always double stranded.
977
The Authors also investigate the effect on molecular weight of heat deg-
978
radation (100°C along 15 minutes in a 0.1 M NaCl solution) applied to frag-
979
ments obtained by previous gamma ray degradation. Their data shows this
980
thermal treatment yields a decrease in molecular weight, but the Authors un-
981
derline that quantitative appreciation of the entity of molecular weight de-
982
crease and of number of fractures is made difficult by the “the known tendency
983
for the polynucleotide chains to remain entangled, in spite of the removal of hydrogen
984
bonding, and for separated chains to re-aggregate on cooling”.
985
A kinetic statistical model for random degradation of a two-stranded pol-
986
ymer model specifically devised by Applequist [10] to analyze DNA degrada-
987
tion provides a more detailed description of the kinetics of double fracture
988
proposed by Peacocke and Preston which accounts for independent probabil-
989
ities of intrachain fractures (termed P-bonds by the Author and denoted in our
990
synthetic review ) and crosslinking hydrogen bonds (H-bonds).
991
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Denoting by the fraction of broken P-bonds, by the fraction of broken
992
H-bonds, and by  the probability that a pair of facing nucleotides is followed
993
by a double chain break, they compute that at the beginning of the degrada-
994
tion process when almost all P-bonds are unbroken the relation holds:
995


(12)
996
This relation is a more explicit determination of the proportionality rela-
997
tion highlighted by Peacocke and Preston and contained in (9). By further in-
998
troducing the simplest assumption that P-bonds are broken by a first-order
999
rate process, viz.  corresponding in the early stages of
1000
degradation when  to the linearized law , they
1001
find that the linear relation:
1002



(13)
1003
excellently fits the asymptotic trend in experimental data  obtained
1004
from enzymatic degradation [54], acidic degradation [55] and thermal degra-
1005
dation (Doty, Marmur, Eigner and Schildkraut, 1960) [12]. The data for ther-
1006
mal degradation is reported together with the fit provided by (13) (divided by
1007
) in Figs. 4(a) and 4(b), respectively.
1008
1009
1010
(a)
(b)
Figure 4. (a) thermal degradation of diplococcus pneumoniae DNA from Doty et al.[12]. (b)
1011
same data represented in the format of Eq. (13) and corresponding least-square linear fit.
1012
1013
This very close fit constitutes a strong proof that the DNA remains actually
1014
two-stranded during the thermal degradation process reported by Doty et al.,
1015
(1960) [12]. This observation is pivotal for the focus of our study for three rea-
1016
sons:
1017
1018
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1) Fragmentation rules out also the plausibility of the 100°C stable molec-
1019
ular weight claim.
1020
2) Doty et al., (1960) [12], based on the circumstance that one of their
1021
determination of the molecular weight of the DNA heated at 84° is
1022
approximately decreased by a factor of two (from 10.5 million to 5.0
1023
million), advance the claim that with aggregation eliminated and depol-
1024
ymerization taken into account it can be said that strand separation did occur
1025
in the very early stages of the exposure to the elevated temperature. Con-
1026
versely, the fit in Fig. 4(b) shows the two-stranded character of the
1027
heated DNA and points to the clear evidence that the continuous fall
1028
of molecular weight is the result of progressive fragmentation into
1029
pieces that substantially remain two-stranded, so that the molecular
1030
weight halving recorded by Doty et al. is only fortuitously in agree-
1031
ment with the value of ½. In this respect it is worth acknowledging
1032
that also Doty et al. [12] in the statement above mention the necessity
1033
to account for “depolymerization“, thus duly excluding the possibility
1034
that halving of molecular might correspond to separation of intact sin-
1035
gle strands:.
1036
3) The elucidation of the substantial two-stranded character of the mate-
1037
rial produced by heating, besides confuting the 100°C dissociation
1038
claim as advanced by Doty et al. in the same paper [12], rules out any
1039
meaningfulness of heating denaturation for the purpose of strand sep-
1040
aration.
1041
1042
A second important highlight in the contribution by Applequist is the role
1043
of the initial induction relatable to the presence of an initially uniform distri-
1044
bution of molecular weights and the determination based on the data by Shu-
1045
maker et al. of the presence of initially broken P-bonds corresponding to
1046
. This numerical determination will be recalled in the discussion
1047
of Section 4.2.
1048
An examination of the type of intra-strand bond breakage is reported in
1049
Section 4 based on a closer review of the insightful analysis by Dekker and
1050
Schachmann (1954).
1051
1052
1053
2.3.9. Evidence from light scattering of DNA fragmentation upon heating
1054
1055
This subsection reviews determinations of DNA degration by heating via
1056
light-scattering experiments reported in publications other than those by Doty
1057
and Rice of 1955 and 1957, which are instead more closely reviewed in the
1058
dedicated Section 3.
1059
Peacocke and Preston (1958) [50] also perform light scattering measure-
1060
ments on DNA degraded by combined exposure to gamma rays and heating
1061
at 100°C for 15 minutes. They confirm observation of a decrease in molecular
1062
weight but highlight that the observation of the effect of heating degradation
1063
in larger DNA molecules is less pronounced and emphasize that the interpre-
1064
tation of the entity of this decrease is affected by the known tendency for the
1065
polynucleotide chains to remains entangled, in spite of the removal of hydrogen bond-
1066
ing, and for separated chains to re-aggregate on cooling”.
1067
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1068
Sadron performed measurements of molecular weights by light scatter-
1069
ing after heating alone (in absence of gamma-rays exposure) and reported his
1070
findings in several publications between 1955 and 1959 [51], [60], [61]. In the
1071
1955 biochemistry congress proceedings (see addendum on page 134) he re-
1072
ports the effect of heating at 100°C along 15 minutes on two DNA prepara-
1073
tions of initial molecular weight . The weights he determines
1074
by light scattering on heated DNA at different NaCl concentrations show that
1075
DNA weight is always reduced as a confirmation that DNA is the more de-
1076
graded the less is the NaCl content.
1077
In particular, the table in his 1955 addendum on page 134 in [51] reports that
1078
just 15 minutes heating in absence of salt bring the molecular weight from
1079
 to  and . In just one experiment with 1M NaCl
1080
he finds, in one of the two preparations, a weight increment to an average
1081
value . Later, Freund, Pouyet and Sadron (1958) [61] report
1082
tests of calf thymus DNA (CV71) in 1M NaCl solutions and obtain by light
1083
scattering for the unheated preparation molecular weight  .
1084
They measure by light scattering the molecular weight in a 0.01 M NaCl
1085
solution after thermal degradation produced by heating at temperatures of
1086
75°, 80°, 85° and 98°C during a variable time . They find that by heating at
1087
temperatures of 80°, 85° and 98°C the ratio  as function of first
1088
increases, reaches a maximum and then drops to a residual value
1089
corresponding to . The point data in the original fig-
1090
ure have been digitized and those corresponding to temperatures of 85°C and
1091
98°C are reported in Fig.5. The only correction applied to the digitized num-
1092
ber was that minutes that were rounded to closest simple fractions of the hour
1093
of multiples of 5 minutes and to ordinates reached at the horizontal asymp-
1094
tote which were rounded to the value 0.047 explicitly reported by Freund et
1095
al.
1096
The Authors find that the higher is the heating temperature the higher is
1097
the maximum, as shown in Fig. 5. This figure shows that heating at 98° brings
1098
this maximum above 1.4 and then makes suddenly drop at approximately
1099
10 minutes to a value below 0.35. It is also seen that at 15 minutes the inter-
1100
polation reaches 10% of the native weight. This measure brings further quan-
1101
titative straightforward evidence of the sudden fragmentation that the DNA
1102
molecule undergoes when heated at temperatures above 90°C.
1103
1104
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1105
Figure 5. Digitized reproduction of Fig. 1 by Freund, Pouyet and Sadron
1106
(1958) [61]. Ratio  vs. heating time . Only data for 98°C and 85°C
1107
are reported.
1108
1109
1110
2.3.10. DNA Heating experiments in presence of different electrolytes
1111
1112
Hamaguchi and Geiduschek (1962) [56] perform a broad analysis of ther-
1113
mal stability of DNA in aqueous solution in presence of many different elec-
1114
trolytes and under many different pH conditions and temperature ranges.
1115
They confirm that thermal stability of DNA has a relatively broad maximum
1116
at pH 7-8 in 0.1 M NaCl and that in any concentration of any of the many
1117
electrolytes therein investigated the upper bounds of the temperature thresh-
1118
old of thermal stability can be never above 92.6°C (see Table I and Fig. 4
1119
therein), irrespective of the electrolytes content and type.
1120
1121
3. Review of the experiments on DNA heating by Doty and Rice (1955,
1122
1957)
1123
1124
The arguments relevant to the effects of DNA heating, and their interpre-
1125
tation, contained in the 1955 short communication by Doty and Rice “The de-
1126
naturation of desoxypentose nucleic acid” [11] deserve the utmost attention
1127
since, to the authors’ knowledge, this paper is chronologically the first record
1128
where it can be read of the 100°C stable molecular weight claim for DNA. This is
1129
in neat disagreement with the big picture consistently emerging from the
1130
studies analyzed in Sections 2.2, about DNA undergoing thermal depolymer-
1131
ization when the threshold of 81°C is surpassed, or even at lower tempera-
1132
tures in less stable conditions. In [11] it is reported indeed that, after heating a
1133
neutral saline solution to 100°C for 15 minutes the product is found to have
1134
the same molecular weight as the native DNA”.
1135
This third section enucleates the experimental evidence reported in [11]
1136
relevant to the effect of DNA heating and contains an analysis of the interpre-
1137
tation given by Doty and Rice to such experimental evidence aimed at under-
1138
standing if, and how, new settling measurements are reported by Doty and
1139
Rice in this 1955 study able to reverse the previous overall experimental-
1140
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theoretical picture on the phenomenon of DNA fragmentation on heating
1141
above 80°.
1142
A critical scientific review is also contextually carried out of results and
1143
interpretations by Doty and Rice following the contemporary scientific debate
1144
on the consequences of DNA heating. To this end important contributions by
1145
K.V. Shooter, R.H. Pain, J.A.V. Butler, P. Alexander, K.A. Stacey and C. Sadron
1146
are also examined in this chapter in more detail.
1147
As [11] is a preliminary note, it is incomplete of exhaustive information
1148
on relevant materials and methods. The lacking information can be however
1149
sourced from the subsequent 1957 publication by Rice and Doty [23] referred
1150
as the continuation of such preliminary note. By joining the information con-
1151
tained in [23] and [11] it is known that the tested samples are of calf thymus
1152
DNA and that the results reported in [11] are obtained from the sample des-
1153
ignated in [23] as SB-11 “prepared according to Simmons Method B”.
1154
The Authors inform that the relevant “preparative procedure involves mul-
1155
tiple extractions of minced thymus in saline-citrate (0.015 M sodium citrate and 0.15
1156
M NaCl) by blending and centrifuging to obtain a sediment containing the nucleo-
1157
protein. Notably, the Authors specify that 30% sodium p-xylene sulfonate is
1158
used to deproteinize the nucleoprotein.
1159
The three fundamental measurements employed by Doty and Rice for
1160
determining the molecular weight are the following:
1161
- sedimentation measurements by ultracentrifuge;
1162
- viscosity measurements by a capillary viscometer;
1163
- light scattering measurements.
1164
1165
Evaluation of molecular weight from this combined sedimentation and
1166
viscosity measurements by Doty and Rice is examined first in Subsection 3.1.
1167
Next, determinations of molecular weight from light scattering measure-
1168
ments by the same Authors are examined in subsection 3.2.
1169
1170
3.1. Molecular weight from combined sedimentation/viscosity measure-
1171
ments and the scientific debate among Doty, Shooter and coworkers
1172
The data in [11] which Doty and Rice report in possible support the 10 stable
1173
molecular weight claim are collected in Table 2.
1174
Table 2. Experimental sedimentation/viscosity measurements reported by Doty and Rice (1955) [11]
1175
employable to infer molecular weight in native and heated DNA. Data relevant to preparation coded
1176
SB-11” in [23].
1177
Quantity
Symbol
Units of
measurement
Value measured
on native DNA
Value measured on
DNA after heating for
15 minutes to 100°
Reduced intrinsic
Viscosity
[]
[
]
72.0
4.3
Sedimentation constant
referred to 20° and
extrapolated to null
dilution coefficient

: Svedberg units
[seconds per 10-13]
21.0
30.0
1178
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Concerning quantities in Table 2 and their units it is worth recalling what
1179
follows.
1180
Quantity [] in Table 2 is the intrinsic viscosity: the specific increase in
1181
relative viscosity, , determined by adding a quantity of DNA associated
1182
with a concentration increment  to the solvent (of viscosity ), divided by
1183
. Accordingly, 
 and, as  is in grams per deciliter, [] is in recip-
1184
rocal units of deciliters/gram. The solvent of Doty and Rice is a saline-citrate
1185
solvent (0.015 M sodium citrate and 0.15 M NaCl).
1186
The sedimentation constant 
is a rate of sedimentation measured by
1187
the ultracentrifuge experiment methodology developed by Svedberg [57]. In-
1188
dicating by the radial coordinate in the axial symmetric setup of the ultra-
1189
centrifuge, the centripetal acceleration is a radial field varying as ,
1190
where is the angular velocity. The rate 
 is measured by an optical system.
1191
through so called schlieren photography which essentially returns diagrams of
1192
changes in refractive index that typically have a «striped» pattern (“schlie-
1193
ren” = strip from German) and that inform about the change in concentration
1194
as function of time and radial coordinate . If the centrifugal field is strong
1195
enough to cause the molecules or particles to sediment with measurable ve-
1196
locity, 
, the sedimentation constant is then

.
1197
The principle of measurement in centrifuge is that the rate of sedimenta-
1198
tion increases with the mass of the particle and with the intensity of accelera-
1199
tion. Tracing a parallel with a falling object of mass immersed in atmos-
1200
phere and in a uniform gravitational field, it is known that due to the presence
1201
of air friction, the object deviates from the Galilean prediction of uniformly
1202
accelerated motion with gravitational acceleration and reaches after a tran-
1203
sitory time a steady velocity. The frictional force , directed upwards, in a
1204
simplest description, is proportional to the velocity of the falling object so
1205
that . As theoretically predicted by the force balance (gravitational
1206
force = frictional force; ; ) and empirically confirmed, the fi-
1207
nal velocity reached by the object increases with the object mass and with
1208
acceleration which for the gravitational field is uniform in space and equal
1209
to the terrestrial gravitational acceleration constant : . The mass is ac-
1210
cordingly computed as 
or 
. As recalled, the main difference
1211
between the acceleration field of the centrifuge set-up and the gravitational
1212
field is that rotation implies a circular relative motion field with acceleration
1213
increasing with the radius by  so that acceleration is no longer uni-
1214
form as in the example of the falling object. The DNA molecules, heavier than
1215
the solvent, by inertia, will appear in the non-inertial rotor reference frame to
1216
be subject to a radial motion component directed towards the periphery of
1217
the centrifuge. This motion component may be causally interpreted as the
1218
consequence of not being sufficiently «pushed» in centripetal direction by the
1219
pressure gradient field that is generated in the solvent in response to the cen-
1220
tripetal acceleration generated by the rotor. Through a special focus lenses
1221
measurements of the concentration along the radial direction are obtained
1222
from measurements of light absorption changes and/or refraction changes.
1223
Records are taken of the «strips» produced by the different optical properties
1224
of this moving «solute clouds». From these striped diagrams it is possible to
1225
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measure concentration changes along the radius and, in turn, evaluate the
1226
speed . The higher the 
 rate of sedimentation of this «dust», at the
1227
same acceleration (and therefore at the same angular velocity of ro-
1228
tation and radial distance in the blender), the greater is the mass of the
1229
DNA molecule or molecule fragment by the proportion 
. The final
1230
raw quantitative measurement obtained from this experiment is the sedimen-
1231
tation constant

 whereby the direct proportionality holds .
1232
Experiments are performed to extrapolate the limit of at zero concentra-
1233
tion (zero superscript), so that direct interaction between molecules can be
1234
excluded. If account is also taken of temperature which can change the sol-
1235
vent viscosity and a reference temperature of 20°C is employed, the symbol
1236

is used to refer to such quantity. Ultracentrifuge sedimentation analysis
1237
of DNA containing a detailed report of observed sedimentation boundaries
1238
are presented by Cecil and Ogston (1948) [57,58]. Also, to the authors’
1239
knowledge, the first reported molecular weight measurements for DNA,
1240
based on the application of combined viscosity-sedimentation methods, are
1241
by Krejci, Sweeny and Hambleton (1949) [59].
1242
1243
Concerning the ultracentrifuge sedimentation experiments, Rice and
1244
Doty [23] report two important information:
1245
When DNA solutions are exposed to 100°C for 15 minutes the hyper-
1246
sharp sedimentation profile is lost and “two differently sedimenting species
1247
in solutions appear.
1248
Such a polydisperse character of the heated solution, in the same words
1249
as Rice and Doty, “posed a difficult problem” and induced the Authors to
1250
erroneously attribute a sedimentation constant of 30  In fact they also
1251
report, Dr. K. V. Shooter and Professor J. A. V. Butler have examined our
1252
sample SB-11 by ultraviolet optics in the ultracentrifuge and found that the dis-
1253
tribution of sedimentation coefficients broadened but remained single-peaked at
1254
21 . when heated at 100°C for 15 min. at a concentration of 10 mg./dl.(see
1255
Shooter et al. [9]).
1256
1257
1258
The scientific debate between Doty and Rice and Shooter et al. deserves
1259
attention. Measurements by Shooter et al. [9] are conveniently reported in Ta-
1260
ble 3 since the Authors re-execute both sedimentation and viscosity measure-
1261
ments for sample SB-11 (in the acknowledgement the donation by Prof. Paul
1262
Doty is mentioned) investigating the effect of 15 mins heating at 100°C and
1263
achieve increased accuracy for sedimentation measurements. Increased accu-
1264
racy is achieved since the Authors employ a preferable lower 0.01% concen-
1265
tration instead of 0.035% of the samples examined in [23], and operate optical
1266
measurements in the ultra-violet range where they had shown that heteroge-
1267
neity with respect to sedimentation coefficient is more easily observed [62]).
1268
By comparing Tables 2 and 3 it is seen that reduced intrinsic viscosity values
1269
are the same in [23] and [62] while a value of 20.0 is more accurately meas-
1270
ured for the sedimentation constant after heating thus identifying and
1271
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correcting the experimental error recognized by Doty and Rice and recalled
1272
above.
1273
1274
Table 3. Sedimentation/viscosity measurements by Shooter et al. [9] in native and heated samples of
1275
0.01% DNA preparation SB-11. Units are the same of Table 2.
1276
Quantity
Value measured
on native DNA
Value measured
on DNA after
heating for 15
minutes to 100°
Reduced intrinsic
Viscosity
[]
72.0
4.3
Sedimentation
constant referred to
20° and extrapolated
to null dilution
coefficient

20.80
20.0
1277
1278
Shooter et al. [9] finalize their study on the effect of DNA heating and
1279
their countercheck of Doty and Rice measurements by concluding that The
1280
results given suggest that in all cases the samples of DNA are degraded on heating
1281
but that the extent of the degradation and the distribution of size and shape of the
1282
fragments produced depends upon the way in which the DNA has been prepared”. It
1283
is important to remark that this conclusion is achieved in [9] upon testing
1284
sedimentation constants and viscosities of four DNA preparations (TNA 7,
1285
TNA 15 , TNA 23 and SB-11) under multiple concentrations, in presence and
1286
in absence of the protective effect of NaCl . The Authors emphasize that sed-
1287
imentation constant decreases in all samples except for samples TNA16 and
1288
SB-11 for which it remains unchanged. They remark that despite the unchanged
1289
sedimentation constant the apparent molecular weight is reduced in all cases. They
1290
have the merit of highlighting the possible fallacies in deducing the molecular
1291
weight from combined sedimentation and viscosity measurements. Fallacies
1292
may arise from reaggregation of the fragments when heating is performed at
1293
higher concentrations. Indeed, in one case they find that heating the TNA23
1294
solution at 0.05% concentration at 100°C for 15 minutes makes the sedimen-
1295
tation constant increase from 27 to 36 Svedberg units. This measurement
1296
shows that particles appear to have possibly even increased their mass, an
1297
effect that may be explained only by reaggregation. They also observe for all
1298
DNA preparations that the “spread of sedimentation coefficients is changed, there
1299
being an increase of material with both high and low sedimentation coefficients”.
1300
Such result is readily interpreted as evidence of the production by 15 minutes
1301
heating of both fragments of lower molecular weight as well as of aggrega-
1302
tions of fragments of higher molecular weight.
1303
Shooter et al. highlight that preparations TNA 23 and SB-11 are obtained, in
1304
particular, by detergent methods (it is worth recalling that SB-11, donated by
1305
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Doty, has been prepared employing sodium p-xylene sulfonate) pointing to the
1306
risk that higher sedimentation constants may be preparation artefacts due to
1307
the use of detergent methods while lower sedimentation constants might be
1308
artefacts due to purification procedures employing enzyme methods which
1309
have a depolymerizing effect.
1310
Next, in their 1957 publication [23] containing the reply to Shooter et al.
1311
countercheck, Rice and Doty do not make considerations on the evidence that
1312
fragments reaggregation readily explains the increase in sedimentation con-
1313
stant from 21 to 30 of Table 2 that they had published in their 1955 pre-
1314
liminary note. On the other hand, Shooter et al. had shown that heating at
1315
0.05% concentration, a value close to the 0.035% concentration employed by
1316
Rice and Doty, makes the sedimentation constant of TNA 23 increase from 27
1317
to 36 .
1318
1319
1320
Doty and Rice reference the empirically well-established relation of
1321
Krigbaum, Flory, Mandelkern and Sheraga [11] which is the following:
1322
1323

,
(14)
1324
In Equation (14) is a universal constant which has the same value irre-
1325
spective of molecular weight, temperature and solvent and depends only on
1326
the flexibility of the molecular chain. Its value ranges between  
1327
 for a flexible coiled molecule and   for ellipsoids of in-
1328
creasing axial ratio, is the solvent viscosity, is Avogadro's number, is
1329
the density of the solution and  is the partial specific volume. All quantities
1330
on the right hand side of Eq. (14) are constants, or can be treated as such, in
1331
the experiments under examination.
1332
1333
Equation (14) permits a more accurate determination of molecular mass.
1334
It can be applied to examine Doty and Rice data of Table 2 and to investigate
1335
the scientific substantiation of their key argument: i.e., to investigate whether
1336
changes induced by 15 mins heating at 100° can be explained exclusively by
1337
calling into question a possible coiling or change in the molecule flexibility,
1338
excluding any molecular weight change, or if compelling evidence exists of
1339
molecular fragmentation from data of Tables 2 and 3.
1340
Subscripts N and H are used below in a way <