Detection of YMDD motif mutants by oligonucleotide chips in lamivudine-untreated patients with chronic hepatitis B virus infection.
ABSTRACT Lamivudine, a nucleoside analogue, has been used widely as an effective antiviral agent for the treatment of patients with chronic hepatitis B virus (HBV) infection. However, the YMDD motif mutation of HBV polymerase resistant to lamivudine occurs very frequently after long term therapy. We developed an oligonucleotide chip for the detection of YMDD motif mutants resistant to lamivudine and investigated the prevalence of the mutants in patients with chronic HBV infection who had not been treated by lamivudine before. Forty patients who had not been treated with lamivudine were included in this study. Serum samples were tested by the oligonucleotide chips designed for detection of wild-type YMDD motif, M552V and M552I. Samples were confirmed by restriction fragment length polymorphism (RFLP) and direct sequencing. M552I mutants were detected by the oligonucleotide chips in 7.5% (3/40) of chronic HBV infected patients (2 chronic hepatitis and 1 cirrhosis). The results were in accordance with those of RFLP. YMDD motif mutants occur as natural genome variabilities in patients with chronic HBV infection who had not been treated with lamivudine before. Oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of mutants resistant to antiviral therapy in chronic HBV infection.
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ABSTRACT: One-year lamivudine therapy significantly suppressed hepatitis B virus (HBV) replication, improved hepatic necroinflammatory activity, and prevented progression of fibrosis. However, the effects of prolonged therapy are unknown. A total of 334 Asian patients with chronic hepatitis B from a previously reported 1-year study were randomized to receive either lamivudine (100 or 25 mg) or placebo for another year. The effects of treatment on serum HBV-DNA suppression, alanine transaminase (ALT) normalization, and hepatitis B e antigen (HBeAg) seroconversion were measured. The presence of YMDD variant HBV and its effect were also determined. A significantly greater proportion of patients achieved sustained HBV-DNA suppression and ALT normalization with 100 mg lamivudine daily for 2 years compared with lamivudine for 1 year followed by placebo for the second year (P<0.001). Daily lamivudine therapy for 2 years was safe and resulted in incremental HBeAg seroconversion from 17% at week 52 to 27% at week 104. HBeAg seroconversion during continued lamivudine therapy increased linearly with increasing pretherapy ALT levels (P< 0.001). Despite the emergence of YMDD mutant in 38% of the patients, they continued to clear serum HBeAg and maintain lower median serum HBV-DNA and ALT levels than baseline values. In contrast, ALT levels increased 8-12 weeks after switching from lamivudine to placebo, but returned to normal once lamivudine treatment was resumed. Treatment with lamivudine for 2 years is both well tolerated and efficacious in patients with chronic hepatitis B.Gastroenterology 08/2000; 119(1):172-80. · 12.82 Impact Factor
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ABSTRACT: Lamivudine therapy induces improvements in chronic hepatitis B in a high proportion of patients, but prolonged therapy is limited by the development of viral resistance. We analyzed clinical responses and virologic resistance in 27 patients treated continuously with lamivudine for 2 to 4 years. Serum transaminases, hepatitis B virus (HBV) DNA by both branched DNA (bDNA) signal amplification and quantitative polymerase chain reaction were monitored at 4- to 8-week intervals. Virologic resistance to lamivudine was confirmed by the presence of mutations in the YMDD motif of the polymerase gene by restriction fragment-length polymorphism analysis. Serum HBV-DNA levels decreased rapidly in all treated patients, falling by 4 to 5 logs within 1 year. Transaminase levels also decreased and were normal in 70% of patients at 1 year, at which point liver histology had improved in 81% of patients. Viral resistance began to emerge after 8 months of therapy, eventually developing in 14 patients, including 76% of hepatitis B e antigen (HBeAg)-positive patients but only 10% of HBeAg-negative patients. Lamivudine withdrawal led to reappearance of wild-type HBV species, but retreatment led to more rapid reappearance of the mutant virus. Clinical, serum biochemical, and histologic improvements were maintained in the 13 patients who did not develop resistance. Thus, long-term therapy with lamivudine resulted in maintained improvements in virologic, biochemical, and histologic features of disease in most patients with HBeAg-negative chronic hepatitis B and in the subgroup of HBeAg-positive patients with high serum transaminase levels. A high rate of resistance limited efficacy, particularly in patients who remained HBeAg positive on therapy.Hepatology 11/2000; 32(4 Pt 1):828-34. · 12.00 Impact Factor
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ABSTRACT: Treatment of hepatitis B virus (HBV) with lamivudine is effective in suppressing virus replication and results in reduced inflammatory activity. However, the emergence of lamivudine-resistant mutant virus, with amino acid substitution in the YMDD motif of DNA polymerase, has been reported. We report the emergence and takeover of YMDD mutant and re-takeover by wild type during and after long-term lamivudine therapy. YMDD mutants were detected in five patients who showed DNA breakthrough (HBV DNA becoming detectable after a period of DNA negativity), which occurred after 9 to 14 months of lamivudine therapy. Four of five mutants had amino acid sequence YIDD, and the remaining mutant had YVDD. Patients with high HBV-DNA titer and/or hepatitis B e antigen tended to develop breakthrough (P = .038). Using a sensitive and specific polymerase chain reaction (PCR)-based method developed in this study, the emergence of YMDD mutants was detected 1 to 4 months before DNA breakthrough, but not detected in any of the pretreatment sera. The mutants were predominant at breakthrough, but were replaced by wild-type virus 3 to 4 months after cessation of therapy in the two patients who discontinued therapy. One of these patients had a relapse of hepatitis. Mutant continued to replicate in the remaining three patients who continued to receive treatment, and relapse occurred in only one of these patients. Our results suggest that the replication of YMDD mutant viruses is less than wild type and is re-overtaken by wild type after cessation of therapy. Re-administration of lamivudine, possibly combined with other antiviral therapy, might be useful in some patients experiencing hepatitis with lamivudine-resistant variants.Hepatology 05/1998; 27(6):1711 - 1716. · 12.00 Impact Factor
Lamivudine, a nucleoside analogue, has been known to be
an effective antiviral agent for hepatitis B virus (HBV) infec-
tion.The effectiveness of lamivudine therapy is indicated by
serum HBV DNA clearance and the seroconversion of HBeAg
to anti-HBe. According to follow-up reports of the multi-
center study, seroconversion rates increased with the dura-
tion of treatment from 17% at 1 yr to 27% and 40% at 2
and 3 yr, respectively (1, 2). However, the emergence of viral
mutants resistant to lamivudine is the main concern with
the treatment. The resistance begins to occur after 8 months
of treatment (3, 4) and the resistance rates increase with the
duration of therapy from 17% at 1 yr to 39% and 57% at 2
and 3 yr, respectively (2). The lamivudine-resistant mutants
affect the YMDD motif of the catalytic domain of the HBV
DNA polymerase. The most common mutants have a change
at codon 552 from M to V (M552V) or from M to I (M552I).
Although the mutations of YMDD motif are thought to be
secondary to lamivudine use, it is considered that the pre-
treatmentmutants already exist in endemic regions like Korea
(5). Oligonucleotide chip technology is a very useful tool for
the rapid and accurate identification of pathogenic microbes
and the detection of drug-resistant and point mutations. We
developed an oligonucleotide chip for the detection of mutants
resistant to lamivudine. This study aimed to determine the
prevalence of natural mutants of YMDD motif by oligonu-
cleotidechip technology in patients with chronic HBV infec-
tion who had never received treatment of any antiviral agent
MATERIALS AND METHODS
This study consisted of 40 patients with chronic HBV
infection who had never received any antiviral medication
including lamivudine. Their average age was 29 yr (from
13 to 57 yr), and there were 38 males and 2 females. Thir-
ty-four patients had chronic hepatitis and 6 had cirrhosis.
HBV DNA purification and target DNA preparation
As soon as blood samples were collected they were refrig-
erated for one hour and left to coagulate, which then were
Jeong Heo, Mong Cho, Hyung Hoi Kim*,
Young Min Shin, Hyun Jung Jang
Hee Kyung Park
Gwang Ha Kim, Dae Hwan Kang,
Geun Am Song, Ung Suk Yang
�, Cheol Min Kim
Department of Internal Medicine, and *Department of
Laboratory Medicine, Pusan National University
College of Medicine; �Department of Microbiology,
Pusan National University College of Natural Science;
�Department of Biochemistry, Pusan National
University College of Medicine, Busan, Korea
Address for correspondence
Mong Cho, M.D.
Department of Internal Medicine, Pusan National
University College of Medicine, 1-10 Ami-dong,
Seo-gu, Busan 602-739, Korea
Tel : +82.51-240-7215, Fax : +82.51-244-8180
E-mail : firstname.lastname@example.org
J Korean Med Sci 2004; 19: 541-6
Copyright � The Korean Academy
of Medical Sciences
Detection of YMDD Motif Mutants by Oligonucleotide Chips in
Lamivudine-Untreated Patients with Chronic Hepatitis B Virus Infection
Lamivudine, a nucleoside analogue, has been used widely as an effective antiviral
agent for the treatment of patients with chronic hepatitis B virus (HBV) infection.
However, the YMDD motif mutation of HBV polymerase resistant to lamivudine
occurs very frequently after long term therapy. We developed an oligonucleotide
chip for the detection of YMDD motif mutants resistant to lamivudine and investi-
gated the prevalence of the mutants in patients with chronic HBV infection who
had not been treated by lamivudine before. Forty patients who had not been treat-
ed with lamivudine were included in this study. Serum samples were tested by the
oligonucleotide chips designed for detection of wild-type YMDD motif, M552V and
M552I. Samples were confirmed by restriction fragment length polymorphism
(RFLP) and direct sequencing. M552I mutants were detected by the oligonucleotide
chips in 7.5% (3/40) of chronic HBV infected patients (2 chronic hepatitis and 1 cir-
rhosis). The results were in accordance with those of RFLP. YMDD motif mutants
occur as natural genome variabilities in patients with chronic HBV infection who
had not been treated with lamivudine before. Oligonucleotide chip technology is a
reliable and useful diagnostic tool for the detection of mutants resistant to antiviral
therapy in chronic HBV infection.
Key Words : Hepatitis, Viral; Hepatitis B, Chronic; Lamivudine; Polymorphism (Genetics); YMDD Motif
Mutants; Oligonucleotide Array Sequence Analysis
Received : 11 November 2003
Accepted : 1 April 2004
J. Heo, M. Cho, H.H. Kim, et al.
centrifuged at 3,000 rpm for 5 min to separate the sera. These
sera were stored at -70℃, and HBV DNA was extracted by
alkaline methods. Eight L of 1 M NaOH and 72 L of fresh
defrosted serum were placed in a sterilized 1.5 mL test tube,
which was shaken for 10 sec and then left at 37℃ for one
hour. In this 80 L mixture of serum and NaOH, 0.1 MHCl
of the same amount was added and shaken for 10 sec, which
was then centrifuged at 12,000 rpm for 10 min. The super-
natant was transferred to a new test tube and storedat -20℃.
Two L of the DNA isolated from this process was used for
each polymerase chain reaction (PCR).
Amplification of the target DNA to detect YMDD-mutant
HBV was prepared by two biotin-modified primers, HBF2-
biotin and HBR-biotin (Table 1). The primers and probes
used in this study were oligonucleotide manufactured by the
Perkin-Elmer DNA synthesizer (BioBasic, Ontario, Canada).
PCR reactants consisted of 500 mM KCl, 100 mM Tris HCl
(pH 9.0), 1% Triton X-100, 0.2 mM dNTP (dATP, dGTP,
dTTP and dCTP), 1.5 mM MgCl2, 20 pmol primer and 1 U
Taq DNA polymerase (QIAGEN, CA, U.S.A.). This mix-
turewas constituted in total to 25 L by adding 2 L of tem-
plate DNA. It was left to react at 95℃ for 3 min, enough to
denature, followed by 30 cycles of reactions at 95℃ for 1
min, 50℃ for another minute, and at 72℃ for 1 min. The
last cycle was extended for 10 min at 72℃. After these reac-
tions, this PCR product was verified to be 180 bp by elec-
trophoresis with 2% agarose gel, stained with ethidium bro-
mideand visualized on a gel documentation system (gel doc
2000, Bio-Rad, CA, U.S.A.).
The design of oligonucleotide DNA chip
One probe (HB552-WM) was to detect wild-type YMDD
motif and four kinds of probes (HB552-MV, HB552-MI1,
HB552-MI2 and HB552-MI3) were to detect YMDD mutant
on the bases of the HBV DNA polymerase sequence (Table
1). The 5 -end of each oligonucleotide probe was aminated
to increase the efficacy of the immobilization of the oligonu-
cleotideto a glass slide. The probes were diluted to 50 pmol
with 3×SCC (300 mM NaCl, 30 mM Na-citrate, pH 7.0)
solution. These probes were printed in duplex arrays as shown
in Fig. 1, and were adhered to the silylated glass slide (Cel
Associates, Texas, U.S.A.) using the Microarrayer (PLXSYS
7500 SQXL Microarryer, Cartesian Technologies, CA, U.S.A.).
To get rid of unattached probes on the surface, the glass
slide was washed with 0.2% SDS and then with distilled
water twice, followed by sodium borohydride solution (300
mL of PBS, 100 mL of ethanol and 1 g of Na2BH4), and finally
washed out with boiling distilled water. The slide was washed
using 0.2% SDS again and then using distilled water at room
The detection of the mutants using the oligonucleotide
The biotin-labeled target DNA was boiled for 10 min and
annealed at 4℃for 10 min to separate the two strands. Hybri-
dization of 2 L of biotin-labeled ssDNA samples to the oligo-
nucleotide chip was performed in hybridization solution, 3 L
of 20×SSPE, 1.35 L of 22.2 M formamide, 0.5 L of bovine
serum albumin (BSA) and 0.1 L of salmon sperm DNA
containing 0.06 L of diluted Cy3-labeled streptavidin (Amer-
sham Pharmacia Biotech, NJ, U.S.A.). Each sampling was
placed on the slide, the cover slip being applied carefully so
that no air bubble was trapped inside, and was left for reac-
tionat 40℃for 30 min. After removing the cover slip using
2×SSC, the slide was washed out using 2×SSC and 0.2×
SCC solution in this order. The GenePix 4000A (Axon Instru-
ments, CA, U.S.A.), non-confocal laser scanner was used for
oligonucleotide chip analysis (543 nm for excitation of Cy3).
The variable sequences are in bold and italic print. W means wild type.
M means mutant type.
*Characters following W or M mean amino acid type encoded by codon
at mutation region. V, valine; I, isoleucine.
Primer and probe*Sequence
Table 1. Primers and probes used in YMDD motif mutant detec-
Fig. 1. This schematic illustration shows the probe layout for the
17 mer oligonucleotide sequences noted in Table 1 and the inter-
pretative results of oligonucleotide chips for YMDD motif mutation
detection. The oligonucleotide probes are patterned as duplex
arrays. ① Positive is mixture of HB552-WM, HB552-MV, HB552-
MI1, HB552-MI2 and HB552-MI3 probes (Table 1), ② 522WM,
③ 552MI3, ④ 552MI2, ⑤ 552MI1 and ⑥ 552MV indicates HB552-
WM, HB552-MV, HB552-MI1, HB552-MI2 and HB552-MI3 probes,
WildYIDD (I1) YIDD (I2) YIDD (I3)YVDD
Duplex spotted probes
YMDD Motif Mutants in Untreated Patients with Chronic HBV Infection
Restriction fragment length polymorphism (RFLP) of
YIDD (I3) mutant
To confirm the results of the oligonucleotide chip experi-
ment, the partial polymerase gene containing YMDD was
analyzed with the restriction fragment length polymorphism
(RFLP) method and the PCR direct-sequencing.
A PCR reactant solution, which consisted of 500 mM KCl,
100 mM Tris HCl (pH 9.0), 1% Triton X-100, 0.2 mM
dNTP (dATP, dGTP, dTTP and dCTP), 1.5 mM MgCl2,
10 pmol primer (BF108 and BR112, Table 2) (4) and 1 U
Taq DNA polymerase (QIAGEN, CA, U.S.A.), was added
to 2 L of HBV DNA purified by the alkaline method to
make it 25 L in total. This mixture solution was left to dena-
tureat 94℃ for 4 min, followed by 35 cycles of 1 min at 94
℃, 1 min at 58℃ and 1 min and 30 sec at 72℃. For the
last cycle, it was left for 10 min at 72℃. The second PCR
was performed under the same circumstances as the first PCR
using 1 L of the first PCR product, YNSspI and BR109
primer. This product was electrophoresed with 2% agarose
gel to confirm that the PCR product was about 160 bp. Six
L of this confirmed product was added to 5 units of the
restriction enzyme SspI (TaKaRa, Shiga, Japan) and SspI buffer
and left for reaction at 37℃ for 3 hr, which was again con-
firmed by electrophoresis with 4% agarose gel.
RFLP of YVDD mutant
As described above, the PCR was performed with BF107
and TMPpu10I primers (the first PCR) and BF111 and TMA-
paLI primers (the second PCR) (Table 2). This product was
electrophoresed with 2% agarose gel to confirm that the PCR
product was about 190 bp. Six L of this confirmed product
was added to 5 units of the restriction enzyme ApaLI (TaKaRa,
Shiga, Japan) and ApaLI buffer and left for reaction at 37℃
Primer Sequence Direction
5 -TTTCCCCCACTGTTTGGCTTTCA- 711-740
TMPpu10I 5 -CAGACTTGGCCCCCAATACCA-
BF 111 5 -TCGGACGGAAACTGCACTTG-3
TMApaLI 5 -CAGACTTGGCCCCCAATACCA-
Table 2. Primers used in sequencing and enzyme digestion
Fig. 2. Sensitivity and specificity of the detection of the YMDD
mutants using oligonucleotide chip. (A) Detection limit of the
YMDD mutants by oligonucleotide chip, (B) Detection capaci-
ty of mixture of wild-type and YMDD motif mutants. YMDD:
YVDD represents ratios of YMDD and YVDD; YMDD:YIDD3
represents ratios of YMDD and YIDD3.
J. Heo, M. Cho, H.H. Kim, et al.
for 3 hr, which was again confirmed by electrophoresis with
4% agarose gel.
Sequencing and sequence analysis
PCR was performed with BF105 and BR112 primers
(Table 2) under the same condition as target DNA prepara-
tion. The sequence of PCR products was determined with
BF111 and BR109 primers, and using the ABI prism BigDye
Terminator cycle sequencing Ready Reaction Kit (Applied
Biosystem Inc., CA, U.S.A.) in the automated BaseStation-
51 DNA fragment analyzer (MJ Research, CA, U.S.A.).
Sensitivity and specificity of oligonucleotide chip
After serially diluting samples (106-103copies/mL) with
known HBV DNA concentrations by the Cobas Amplicor
HBV Monitor testTM‚ (Roche Diagnostics Systems, Meylan,
France), the detection limit of the DNA chip was evaluated.
The detection limit obtained by a DNA chip assay was 104
copies/mL of serum by the first PCR (Fig. 2A) and 103copies/
mL by nested PCR (data not shown). We constructed plas-
mids containing a copy of the YMDD, YVDD and YIDD
of the polymerase gene of HBV. The YMDD, YVDD and
YIDD plasmids (cloned HBV DNA) were used to deter-
minethe capacity of the DNA chip to detect wild-type HBV
and YMDD motif mutant in a single sample. We could detect
the YMDD motif mutant with the DNA chip when the
mutant was mixed with a ten-fold-higher and -lower amount
of wild-type HBV (Fig. 2B).
Detection of YMDD Mutants by oligonucleotide chip
Thirty seven patients out of 40 had the wild-type YMDD
mutants, and 3 of them (7.5%) had the mixed type of YMDD
and YIDD (I3). Among 3 patients with any mutation (24th,
27th and 37th samples), 2 had chronic hepatitis and one had
liver cirrhosis (Table 3, Fig. 3).
Detection of YMDD mutants by RFLP and direct sequencing
When the codon 552 of the second PCR product by the
TMApaLI primer was GTG (GTGCAC), there was no band
seen, and was 30 bp shorter by the limiting enzyme ApaLI,
throughout the samples. When the codon 552 of the second
PCR product using the YNSspI primer was ATT (AATATT),
about 30 bp were cut by the limiting enzyme SspI, and this
shortened band was confirmed by electrophoresis with 4%
agarose gel. Out of the total samples, shortened bands were
observed in sample Nos. 24, 27 and 37, which were identi-
cal to YIDD mutants (ATT) detected by the oligonucleotide
chip. No shortened band was seen in the other samples (Fig. 4).
The PCR direct-sequencing method was unable to prove
YMDD mutation, which was confirmed by the oligonucleo-
tide chip (Fig. 5).
The YMDD motif mutant of the polymerase gene is fre-
quently found in patients who used lamivudine on a long
term basis, and it is usually reported to emerge after 8 months
of therapy (2, 6, 7). The selection of a mutation in the YMDD
motif is known to occur several months earlier than the phe-
notypic resistance, however the information about how early
the genotypic resistance occurs and what causes the resistance
is limited due to the small number of subjects and differences
in detection methods (3, 4, 8). Hence the rate of hepatitis B
resistance to lamivudine might increase further when more
sensitive tests are used to detect the small amount of YMDD
mutants among the wild type HBV, which would impact
upon detection and monitoring the resistance.
This study demonstrated by oligonucleotide chip technolo-
Patient No. Age (yr) Sex ALT (IU/L) HBeAg Anti-HBe HBV DNA (copies/mL) Diagnosis
Table 3. Clinical features of patients with YMDD motif mutation (YMDD+YIDD) at the time of sampling
Fig. 3. Scanned images of oligonucleotide chip (A) wild type
[YMDD], (B), (C), (D) YMDD motif mutation [YMDD+YIDD(I3)],
slides of No. 24, 27 and 37, respectively.
gy that the YMDD mutants exist naturally in the patients
with chronic hepatitis B infection who had not received lami-
YMDD mutants have a significantly low replication abili-
ty. It is thought that naturally occurring YMDD mutants
occupy very small portion of total HBV, and very sensitive
investigative tools are required to prove it. Oligonucleotide
chip technology is a very useful tool for the rapid and accu-
rate identification of pathogenic microbes and the detection
of drug-resistant and point mutations. This method has such
sensitivity that it can find rifampin-resistant tuberculosis
resultant from point mutation in amounts of less than 1%
(9). Oligonucleotide chip technology has been expected to
be clinically applicable to detect minor variants in viral quasi-
species and to increase our knowledge of spontaneous viral
genome variability and selection of mutants by antiviral thera-
py(10). We developed the oligonucleotide chip for the detec-
tion of mutants resistant to lamivudine. In the experiment
using reference sequences generated by site-directed muta-
genesis, we confirmed the sensitivity and specificity of probes.
The oligonucleotide chips could specifically detect mutations
in the YMDD motif (codon 552) of the HBV DNA poly-
merase.The chips included three types of probe (five species):
wild-probe (YMDD), valine-probe (YVDD) and isoleucine-
probes (YIDD1, YIDD2 and YIDD3). We could accurately
detect YMDD motif mutants of YMDD, YVDD, YIDD2
and YIDD3 types in sera from chronic HBV-infected patients.
These chip results were in accordance with results from RFLP,
sequence analysis and allele-specific PCR. We could detect
easily as little as 10% of coexisting mutant viruses in wild
The oligonucleotide chip method detected 3 YIDD mutants
in the 40 patients included in this study. Only ATT (YIDD3)
was detected in all three samples. This chip result was con-
sistent with that of the RFLP analysis. The mutants were
not detected by direct-sequencing and this is reasonable when
taking into consideration that naturally occurring YMDD
mutants are a minor portion of the total HBV and that the
standard base sequence determinant method requires more
than 25% of mutants among HBV.
Our study supports that oligonucleotide chip technology
may have an important role as a reliable and useful diagnos-
tic tool for the early detection of mutants resistant to lamivu-
1. Liaw YF, Leung NW, Chang TT, Guan R, Tai DI, Ng KY, Chien
RN, Dent J, Roman L, Edmundson S, Lai CL. Effects of extended
lamivudine therapy in Asian patients with chronic hepatitis B. Asia
Hepatitis Lamivudine Study Group. Gastroenterology 2000; 119:
2. Leung NW, Lai CL, Chang TT, Guan R, Lee CM, Ng KY, Lim SG,
YMDD Motif Mutants in Untreated Patients with Chronic HBV Infection
Fig. 4. (A) YVDD RFLP anal-
ysis in 4% agarose gel does
not show any band digest-
ed by the restriction enzyme
ApaLI. M: 100 bp ladder.
(B) YIDD RFLP analysis in
4% agarose gel shows DNA
bands (arrows) digested by
the restriction enzyme SspI
in 3 YIDD (I3) mutants (sam-
ple Nos. 24, 27 and 37). M:
100 bp ladder.
M 1 2 3 . . . . .
23 M 24 27
. . . . .
Digested DNA bands by SspI
No digested band by ApaLI
Fig. 5. The sequences of 290 bp DNA including codon 552 rep-
resent only the wild-type (ATG) in the samples with YIDD mutants
(No. 24, 27 and 37).
Wu PC, Dent JC, Edmundson S, Condreay LD, Chien RN. On behalf
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