Large-scale RT-PCR recovery of full-length cDNA clones.

Abstract

Pseudogenes, alternative transcripts, noncoding RNA, and polymorphisms each add extensive complexity to the mammalian transcriptome and confound estimation of the total number of genes. Despite advanced algorithms for gene prediction and several large-scale efforts to obtain cDNA clones for all human open reading frames (ORFs), no single collection is complete. To enhance this effort, we have developed a high-throughput pipeline for reverse transcription PCR (RT-PCR) gene recovery. Most importantly, novel molecular strategies for improving RT-PCR yield of transcripts that have been difficult to isolate by other means and computational strategies for clone sequence validation have been developed and optimized. This systematic gene recovery pipeline allows both rescue of predicted human and rat genes and provides insight into the complexity of the transcriptome through comparisons with existing data sets.

Full text

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690 BioTechniques Vol. 36, No. 4 (2004)
DRUG DISCOVERY AND GENOMIC TECHNOLOGIES
INTRODUCTION
Direct study of cDNAs has an im-
portant role for the analysis of genome
sequences (1,2). The most significant
contributions for discovering novel
transcripts and precisely defining the
structure of known genes have been
from expressed sequence tag (EST) and
full-length cDNA sequence analysis.
When a cDNA sequence is known with
high confidence, it can also be used for
validating the genomic sequence and for
characterizing alternative gene struc-
tures and heterogeneous transcripts that
differ from known sequences.
There are multiple efforts that aim
to capture the sequence of full-length
clones that can be directly obtained
from the cDNA libraries made from
mammals and other select organisms,
such as zebrafish, Drosophila, and
Caenorhabditis elegans (3–8). Among
these, the Mammalian Gene Collec-
tion (MGC; http://mgc.nci.nih.gov) is
distinguished by a commitment to ex-
tremely high clone and sequence qual-
ity, as well as aiming to rescue at least
one representative transcript from each
human gene that codes for a protein
(9,10). Currently, a nonredundant set of
>10,000 human and >9000 mouse full-
length open reading frame (ORF) clones
have been sequenced and verified by
the MGC program (10). This is an im-
pressive start. However, there are still
a large number of genes missing in the
collection, as it is estimated that there
are 25,000–30,000 protein-coding genes
comprising the mammalian transcrip-
tome. In part, this reflects the chosen
approach of relatively shallow sampling
from multiple clone libraries. It also re-
flects the biological reality that many
genes are unlikely to be found except at
very low levels in obscure cell types.
Many of the cDNA clones missing
from the central collections are at least
partially characterized in different da-
tabases or publications (11,12). There-
fore, we elected to develop scalable
reverse transcription PCR (RT-PCR)-
based methods for cDNA clone rescue
utilizing these existing sequences. A
particular focus has been those clones
that were difficult to recover from pre-
vious large-scale efforts. These pro-
cedures include simplified reliable
PCR primer design, stockpiling RNA
and tissue pools, accurate and robust
96-well format RT-PCR, streamlined
sequencing methods, and custom data
management strategies. From the ini-
tial results, we have observed many
discrepancies between the sequences of
the transcripts that we have recovered
and the corresponding sequences in the
databases, and these lead to interesting
insights into the heterogeneity of the
mammalian transcriptome. Overall,
the ease and efficiency of the RT-PCR
cDNA clone rescue pipeline suggests
that this method could economically
replace traditional cDNA library clon-
ing in any situation in which sufficient
sequence is known to design full-length
PCR primers. Therefore, it is especially
useful for the discovery of novel genes
through gene prediction methods. Es-
tablishing this platform is an impor-
tant step towards the development of a
complete set of reagents representing
the mammalian transcriptome.
MATERIALS AND METHODS
Target Selection and Large-Scale
High-Throughput Primer Design
Target cDNAs, for which there were
no publicly available clones, were ex-
tracted from GenBank® [thanks to
Lukas Wagner, National Center for
Biotechnology Information (NCBI),
National Institutes of Health (NIH)].
Baylor College of Medicine, Houston, TX, USA
Large-scale RT-PCR recovery of full-length
cDNA clones
Jia Qian Wu, Angela M. Garcia, Steven Hulyk, Anna Sneed, Carla Kowis, Ye Yuan, David Steffen,
John D. McPherson, Preethi H. Gunaratne, and Richard A. Gibbs
BioTechniques 36:690-700 (April 3004)
Pseudogenes, alternative transcripts, noncoding RNA, and polymorphisms each add extensive complexity to the mammalian tran-
scriptome and confound estimation of the total number of genes. Despite advanced algorithms for gene prediction and several
large-scale efforts to obtain cDNA clones for all human open reading frames (ORFs), no single collection is complete. To enhance
this effort, we have developed a high-throughput pipeline for reverse transcription PCR (RT-PCR) gene recovery. Most importantly,
novel molecular strategies for improving RT-PCR yield of transcripts that have been difficult to isolate by other means and compu-
tational strategies for clone sequence validation have been developed and optimized. This systematic gene recovery pipeline allows
both rescue of predicted human and rat genes and provides insight into the complexity of the transcriptome through comparisons
with existing data sets.

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Vol. 36, No. 4 (2004) BioTechniques 691
Each of the clones had one form in
public databases, which we download-
ed for local use. Each of the sequences
contains putative complete ORFs, but
not necessarily additional untranslated
region (UTR) sequences.
Algorithms were developed to de-
sign primers within 5′ and 3′ UTR se-
quences in order to amplify the entire
ORF of each gene. When no UTR se-
quences were available, the primers
were positioned at the very beginning
of the ORF in order to include the cod-
ing region if possible.
The algorithm we designed first at-
tempted to select the primers with the
theoretical best characteristics based
on Restricted Criteria, the most strin-
gent requirements in terms of melting
temperature (Tm), GC content, and sec-
ondary structures. If no such primers
were available in this region, the pro-
gram adopted less stringent standards,
or Relaxed Criteria, to select the “next
best” primers. If both attempts failed,
the primer pair was located adjacent to
the border of the ORF with minimum
requirement of sequence composition
based on Forced Criteria. The details of
the design criteria are shown in Table 1.
cDNA Synthesis from the Pooled
Tissues
For use in human cDNA clone res-
cue, equal amounts of total RNA were
pooled from 20 human tissues, includ-
ing adrenal gland, bone marrow, brain
cerebellum, brain (whole), fetal brain,
fetal liver, heart, kidney, liver, lung,
placenta, prostate, salivary gland,
skeletal muscle, spleen, testis, thymus,
thyroid gland, trachea, and uterus (all
from Human Total RNA Master Panel
II; BD Biosciences Clontech, Palo
Alto, CA, USA). A second human to-
tal RNA “supplemental” pool contain-
ing samples from human breast, co-
lon, pancreas, and stomach (all from
Ambion, Austin, TX, USA) was also
used in later experiments. For mouse
cDNA clone rescue, equal amounts
of total RNA were pooled from 15
mouse tissues including an 11-day
embryo, a 15-day embryo, a 17-day
embryo, brain (whole), heart, liver,
lung, prostate, salivary gland, smooth
muscle, spleen, stomach, testis, thy-
mus, uterus (all from Mouse Total
RNA Master Panel; BD Biosciences
Clontech). Reverse transcription reac-
tions (13) were performed in bulk and
then distributed for the subsequent
PCR amplifications. A total of 4 μg
RNA was used in a final volume of 20
μL reverse transcription reaction (200
ng/μL). Reverse transcription reac-
tions were primed by oligo(dT) using
200 U SuperScript™ II Reverse Tran-
scriptase in a 20-μL reaction (Invitro-
gen, Carlsbad, CA, USA). In order to
recover longer transcripts efficiently,
we included the proof reading enzyme
Pfu (5 U) and 10% dimethyl sulfoxide
(DMSO) in the reverse transcription
reaction (14).
Table 1. Primer Design Criteria
Primer Design CriteriaRestricted CriteriaRelaxed CriteriaForced Criteria
Primer location
≥80 bp away from
the ORF
≥80 bp away from
the ORF
Force primers to
flank the ORFa
Whole ORF
YesYesAll or most of the
ORF
GC content
35%–75%30%–75%N.A.
Length
25–30 bp20–30 bp N.A.
Tm range
Hairpin structure
65°–80°C
Avoid HP ≥ 3 bp
60°–80°C
Avoid HP ≥ 4 bp
60°–80°C
N.A.
Homology with other
sequences
AvoidAvoid N.A.
Homopolymeric runs
Avoid ≥ 3 bpAvoid ≥ 4 bpN.A.
GC clamp
Avoid N.A. N.A.
ORF, open reading frame; Tm, melting temperature; HP, hairpin; N.A., not applicable; UTR, untranslated
region.
aPrimers are located 80 bp to the ORF, or position 1 to 27 of the Refseq sequence if 5′ UTR < 80 bp, or
the last 27 bp of the RefSeq sequence if the 3′ UTR < 80 bp.

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692 BioTechniques Vol. 36, No. 4 (2004)
DRUG DISCOVERY AND GENOMIC TECHNOLOGIES
RT-PCR Amplification of Specific
Genes
The reverse transcription reactions
were followed by touchdown PCR
(15) combined with an “autosegment
extension” PCR program. One micro-
liter reverse transcription reaction from
the above was used in 50 μL of PCR.
Annealing temperatures were initially
set at approximately 3°–10°C above
the calculated primer Tm. During the
following 5–7 cycles, the annealing
temperature was reduced by 2°C each
cycle, until it reached a temperature
approximately 2°–5°C below the cal-
culated primer Tm. The rest of the am-
plification cycles use this temperature.
Using the autosegment extension PCR
program, PCR extension times were
extended by 15 s each cycle, starting at
the 15th cycle.
Specific primers were used to am-
plify the entire coding region of each
gene. Twenty-eight cycles of PCR were
performed using first strand cDNA as
template and the Advantage™ 2 PCR
Enzyme System (BD Biosciences Clon-
tech). To reduce mutations introduced
by polymerase misincorporation, the
majority of the PCR products after 28
cycles were separated and saved for
cloning. A small aliquot of the PCR
products (10 μL) was amplified for 35
PCR cycles to produce an adequate
amount of DNA to be visualized on an
agarose gel. Kodak® 1D Image Analy-
sis Software v. 3.0.1 (Eastman Kodak,
Rochester, NY, USA) was used to es-
timate the product sizes. The observed
sizes were compared with the expected
sizes, and data were recorded into a da-
tabase. For genes that failed to yield a
PCR product, RT-PCRs were repeated
either using alternative tissue sources,
by increasing the number of PCR cycles
to 45, or by using redesigned primers.
Cloning PCR Products and
Prescreening for the Inserts Size
and Identity
All PCR products were cloned uti-
lizing the PCR-Script® Amp Cloning
Kit (Stratagene, La Jolla, CA, USA)
as follows. PCR products were puri-
fied with the QIAquick® 96-well PCR
purification kit (Qiagen, Valencia, CA,
USA). The purified PCR products were
then “end-repaired” and inserted into
the PCR-Script Amp cloning vector.
Transformation was performed with
XL10-Gold® Kan ultracompetent cells
(Stratagene) in 96-well format. β-Ga-
lactosidase activity screen was used to
distinguish vectors without insert. A
maximum of 12 white colonies of each
gene were picked for DNA preparation.
The DNA of each subclone was di-
gested with EcoRI and NotI restriction
endonuclease and analyzed by agarose
gel electrophoresis in order to deter-
mine the approximate size of the insert.
The digestion data were captured in a
database established for clone manage-
ment and internal tracking of clones in
the pipeline. Band calling and match-
ing software were integrated to develop
lists of genes and subclones to be passed
onto the next step. Six subclones with
inserts of expected sizes were then end-
sequenced to verify their identity before
attempting full-length sequencing.
Full-Length Sequencing by Primer
Walking
Standard oligonucleotide walk-
ing techniques were used to sequence
multiple subclones obtained from the
RT- PCR procedure (16). Software has
been developed to manage the task of
intelligent sequencing primer design,
and the primers were synthesized based
upon the expected sequence from the
database. Forward and reverse primers
were designed every 350 bp to cover
the entire length of the cDNAs. When a
complete and “acceptable” (see defini-
tion of acceptable in the section entitled
Extensive Transcript Heterogeneity
and Clone Acceptability) subclone was
identified, sequencing on all remain-
ing subclones from that targeted gene
was terminated. The resulting sequence
reads were assembled using the Phred/
Phrap computer programs (17) and
edited via the Consed editor (18). The
known sequence from the target gene
was incorporated to facilitate alignment
of the individual subclone reads during
assembly.
Finishing and Sequence Analysis
After assembly, additional sequenc-
ing and editing were required to ensure
the accuracy of the data. Low-qual-
ity regions or unsure bases were con-
firmed through a minimal number of
primer walks, unless an obvious error
was found within the ORF, in which
case the sequencing of that subclone
was terminated. Each subclone was
sequenced to the standard of an accu-
racy of less than one sequencing error
expected per 10,000 bases.
Final clone acceptability test pro-
grams use the cross-match imple-
mentation of the Smith/Waterman
alignment algorithm (19) to compare
sequences to RefSeq data and the
BLAT program (20) to compare to
the finished human genome sequence
April 2003 freeze (http://genome.
ucsc.edu). The resulting alignments
were analyzed for differences be-
tween our sequences and the reference
sequences. Discrepancies, including
substitution and deletions/insertions,
were recorded automatically into a
database by a computer program. An-
other computer program categorized
subclones as follows: subclones with
nucleotide discrepancies outside of
the ORF or nucleotide discrepancies
that did not lead to amino acid chang-
es were considered as acceptable rep-
resentative cDNA clones. Subclones
with mismatches to the RefSeq se-
quence but that perfectly matched
the reference genome sequence were
also accepted. The clone sequences
were also compared to existing EST,
mRNA, and single-nucleotide poly-
morphism (SNP) databases by the
Human Genome Project team at the
University of California, Santa Cruz
(UCSC). The sequence variations that
were different from the genome se-
quence or RefSeq but were supported
by information from the above data-
bases were acceptable.
RESULTS
We have developed a pipeline for the
rescue of functional cDNA clones from
mammalian genes that were difficult to
recover using other methods. The over-
view for the high-throughput RT-PCR
rescue is shown in Figure 1. The prima-
ry areas developed to enable the pipe-
line are primer design, RT-PCR, clon-
ing, and sequence evaluation. We have
generated more than 570 cDNA clones

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Vol. 36, No. 4 (2004) BioTechniques 693
from humans and mice, and the recov-
ered transcripts show extensive tran-
script heterogeneity when compared to
RefSeq and genomic sequences.
Automated High-Throughput
Primer Design
An automated program we devel-
oped has designed primers for over
10,000 genes. Efficient primer design
at this scale was a significant challenge
at the initiation of this project (see Dis-
cussion). In general, the task of primer
design is a balance between the place-
ment of the primer position relative to
the ORF and the constraints imposed
by the composition of the underlying
sequence. The software is now able to
generate PCR primer designs for 99%
of the candidate sequences. Our prim-
ers were located in the 5′ or 3′ UTR
adjacent to the ORF when possible to
amplify complete ORF (Figure 2A).
The primers were generated using three
different stringency levels of design
criteria resulting in a variation in the
characteristic of primers. As expected,
the restrict/restrict combination ap-
peared to be the most successful. Only
minimal differences were observed,
however, between the efficiencies of
primer pairs produced by other com-
binations of the three-design criteria
(Figure 2B).
Reverse-Transcription Reaction and
PCR Amplification
Our gene rescue targets were those
known genes not recovered via previ-
ous cDNA sequencing efforts. In order
to increase the chance of recovering
these genes, our RNA source was a
mixture of 20 human or 15 mouse tis-
sues, which represented the expression
spectrum of the majority of gene tar-
gets. PCR conditions were optimized to
increase yield in a 96-well format with-
out introducing extra mutations due to
polymerase infidelity. This included
adjusting the concentration of various
reagents, using minimized denatur-
ing times, and varying annealing tem-
perature, extension temperature, and
extension time. Combinations of com-
mercially available reverse transcrip-
tion and PCR enzymes were procured
and tested. The yields ranged from
15% to 50%. We chose SuperScript II
reverse transcriptase and Advantage
2 PCR Enzyme System for their high
yield and relative accuracy. Using these
optimized conditions, RT-PCR was at-
Figure 1. High-throughput RT-PCR gene recovery pipeline. At several points in the pipeline, a fail-
ure invokes an alternative protocol to provide materials to reenter the pipeline. RT-PCR, reverse tran-
scription PCR.

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694 BioTechniques Vol. 36, No. 4 (2004)
DRUG DISCOVERY AND GENOMIC TECHNOLOGIES
tempted on a total of more than 2800
genes, approximately 65% (1874/2889)
of which yielded a PCR product of any
kind, and about 51% (1464/2889) gen-
erated visible products of the expected
size (this is the yield we refer to) when
analyzed using agarose gel electropho-
resis (Figure 3, A and B).
In our initial studies, we observed
a pronounced lowering of the effi-
ciency of the amplification of longer
fragments. Therefore, we sorted tar-
get genes into different size fractions
and established optimal PCR condi-
tions for genes of different lengths. We
were able to amplify transcripts up to
10 kb by adding Pfu and DMSO to the
reverse transcription reaction and by
using longer PCR extension times up
to 6 min. With the tailored conditions,
we found that size has less impact on
the efficiency of recovery. The current
yield for fragments in the 0–1.2 kb
range is approximately 60%, and for
longer fragments up to 10 kb, the value
is approximately 50%.
In an effort to recover additional
clones, experiments focusing on the
RNA source, number of PCR cycles,
and primer redesign were performed.
The genes that were not isolated us-
ing the initial Human Master Panel
RNA source were processed for a
second round of RT-PCR utilizing a
supplemental panel of four tissues
(see Materials and Methods). The ex-
pected products covered the full range
of sizes of 0–3 kb. These tissues were
selected on the basis of the fact that
a large fraction of the PCR-negative
genes in the first round were expected
to be expressed in these four tissues
(data not shown). Approximately 30%
of genes that failed to yield a prod-
uct from the initial RT-PCR attempt
(PCR negative) were rescued through
a second round of PCR using these
alternate tissue sources. Additionally,
increasing PCR to 45 cycles resulted
in a 25%–30% yield among PCR-neg-
ative genes. Finally, a set of 192 PCR-
negative genes, which were expressed
in the initial Human Master Panel
RNA source, were subjected to a sec-
ond round of primer design. The sec-
ond round PCR yield with new prim-
ers was as low as 7%. In summary, the
accumulative yield reached 75% after
the above PCR experiments.
Cloning, Prescreening for Full-
length Sequencing, and Sequence
Evaluation
A major improvement during the
blunt-end PCR cloning procedure was
to convert to a 96-well format. This
Figure 3. Large-scale RT-PCR cDNA recovery. (A) Agarose gel electrophoresis of samples from 96-
well format RT-PCR with the Advantage 2 Enzyme System. (B) Gene size distribution and recovery by
RT-PCR. RT-PCR, reverse transcription PCR.
Figure 2. Primer design. (A) mRNA structure and primer locations. (B) The effect of various combina-
tions of primer design criteria on PCR yield. There are no examples in the restrict/relax or forced/restrict
categories. ORF, open reading frame; UTR, untranslated region.