ISSN: 2320-5407 Int. J. Adv. Res.

9(08), 37-43

Journal Homepage: - www.journalijar.com

Article DOI: 10.21474/IJAR01/13226

DOI URL: http://dx.doi.org/10.21474/IJAR01/13226

RESEARCH ARTICLE
SCREENING OF C-DNA LIBRARY USING COLONY PCR

Sakshi Narayan
……………………………………………………………………………………………………....
Manuscript Info Abstract
……………………. ………………………………………………………………
Manuscript History The principal of cdna strand was that it was combined from complete
Received: 05 June 2021 rna utilizing an oligo(dt)- containing preliminary. After oligo(dg)
Final Accepted: 09 July 2021 following the absolute cdna was enhanced by pcr utilizing two
Published: August 2021 groundworks correlative to oligo(da) and oligo(dg) closures of the cdna
beginning from 10 j558lμm3 myeloma cells, absolute cdna was
incorporated and intensified roughly 10 5 overlay. A library containing
10 6 clones was set up from 1/6 of the enhanced cdna. Screening of the
library with tests for three qualities communicated in these cells
uncovered various comparing clones for each situation. The longest
acquired clones contained supplements of 1. 5kb length. No
arrangements starting from transporters or from rrna was found in 14
haphazardly picked clones.
Copy Right, IJAR, 2021,. All rights reserved.
……………………………………………………………………………………………………....
Introduction:-
The cloning of communicated qualities and the polymerase chain response (PCR), two biotechnological leap forwards of the
1970s and 1980s, keep on assuming huge parts in science today Specifically, cloning includes the combination of DNA
from mRNA utilizing a catalyst called invert transcriptase. Albeit this technique inverts the progression of hereditary data as
depicted by the focal doctrine, it adequately impersonates the cycle by which RNA infections "flip" the bearing of record in
their host cells, accordingly making these phones make viral DNA despite the fact that the actual infections contain just
RNA. Interestingly, the polymerase chain response doesn't include the utilization of an underlying mRNA format to make
DNA. Maybe, PCR includes the blend of various duplicates of explicit DNA sections utilizing a catalyst known as DNA
polymerase. This strategy takes into account the production of in a real sense billions of DNA atoms inside only hours,
making it substantially more effective than the cloning of communicated qualities. In any case, cloning stays the go-to
technique for scientists when just the mRNA layout (and not the DNA format) of an arrangement of interest is accessible

Review Of Literature:-
The experiment on Colony PCR to Approach Screen cDNA Libraries for Full-Length Coding Sequences-cDNA is a
generally utilized, hearty, and reproducible instrument for genome-wide articulation investigation in any life form, without
the requirement for earlier grouping information . To this end, the twofold abandoned cDNA format is processed with
limitation catalysts, trailed by ligation of explicit connectors to the tacky closures of the processed cDNA. Hence, a subset
of the limitation parts is enhanced by PCR by utilizing groundworks with a couple of specific nucleotides notwithstanding
the arrangement correlative to the connector and limitation site successions. To recognize the differentially communicated
qualities, the relating cDNA labels are cleaned from the polyacrylamide gel, reamplified, and sequenced. Notwithstanding,
the subsequent cDNA label successions frequently don't relate to FL-coding arrangements and, along these lines, don't give
adequate grouping data to utilitarian portrayal.

Corresponding Author:- Sakshi Narayan

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for example, a cDNA tag or some other kind of EST, is the fast intensification of cDNA closes colony - PCR methodology,
in which connectors ligated to the 3′ and 5′ finishes of the cDNA are utilized to specifically enhance these 3′ or 5′ cDNA
parts by PCR through a mix of quality explicit and connector explicit preliminaries . A typical issue related with colony -
PCRs is the enhancement of vague PCR items because of the presence of one of the groundwork successions in all cDNAs .
A few techniques have been created to take care of this issue, yet the vast majority of them depend on extra enzymatic
strides after fruition of the primary strand cDNA union, which may present slip-ups . An option in contrast to the colony -
PCR technique is the screening of cDNA libraries . This customary way of library screening is work escalated, particularly
when evaluating for uncommon records . A more quick way to deal with get a clone of interest from a cDNA library is by
PCR screening of pooled clones. This methodology requires showing of individual clones into microtiter wells, and is hence
just pragmatic for bountiful records . The got PCR parts are self-ligated, changed into Escherichia coli and plated. In this
way, singular settlements are picked for DNA seclusion and sequencing . Consequently, in light of the tedious and work
concentrated iterative screening of various cDNA libraries or the requirement for guided procedures to deal with singular
clones in a colony-PCR approach, to acquire the FL cDNA clone of huge quantities of inadequate cDNAs in equal, may end
up being a genuine test.

Methodology:-
A medium-throughput screening system, in light of joining of polymerase chain response (PCR) and province hybridization,
was fostered that permits in equal screening of a cDNA library for FL clones comparing to fragmented cDNAs. The
technique was applied to evaluate for the FL open perusing casings of a choice of 163 cDNA-AFLP labels from three
diverse restorative plants, prompting the distinguishing proof of 109 (67%) FL clones. Moreover, the convention takes into
account the utilization of numerous tests in a solitary hybridization occasion, hence altogether expanding the throughput
when evaluating for uncommon records.

Results And Conclusions:-

The introduced system offers a productive strategy for the transformation of inadequate communicated succession labels
(ESTs, for example, cDNA-AFLP labels, to FL-coding arrangements.

Utilizing Reverse Transcriptase to Clone Expressed C-DNA.

Most cDNA particles that were orchestrated in a solitary response were inadequate, with the 5′ finish of the mRNA not
addressed in the last cDNA.

Ultimately, in the last part of the 1990s, Piero Carninci and his partners at the Genome Science Laboratory in Ibaraki, Japan,
conceived a progression of techniques to get around this and different issues. Specifically, these scientists fostered another
procedure for choosing full-length cDNA atoms. This cycle is known as biotin covering, and it includes covering the 5′
finish of the mRNA with a biotin gathering and afterward washing the cDNAs with a RNA processing protein, similar to
RNAse I. When washed with an answer containing a cap-restricting protein, the entirety of the cDNA-mRNA half breeds
with just halfway cDNA duplicates are out of hand, in this manner just leaving behind the mixtures with full-length cDNA
atoms. Truth be told, the analysts showed that biotin covering yielded about 95% full-length cDNA clones

Today, researchers proceed to construct and use what are known as cDNA libraries, or assortments of cDNAs from specific
tissues accumulated at specific occasions during a living being's life cycle.

Researchers frequently produce cDNA libraries as an approach to discover qualities of interest. They screen these libraries
utilizing what are known as tests - corresponding bits of DNA that hybridize to the cDNA particles. They likewise use
cDNA libraries to distinguish qualities that are communicated diversely in various sorts of tissues or at various formative
stages. Libraries of cDNA atoms give depictions of quality movement, in light of the fact that solitary those qualities that are
really communicated and interpreted into mRNA particles can be cl

cDNA library screening

cDNA libraries depend on mRNA supplements, and address the mRNA cosmetics inside a given cell or tissue. Libraries
give a great deal of data about the character and usefulness of explicit qualities. Libraries likewise give corresponding
experiences into the wealth of RNA created in a given cell or tissue in light of the fact that the more a mRNA is
communicated, the more cDNA will be delivered and the other way around.

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cDNA libraries are not quite the same as genomic libraries in the accompanying manners
mRNA is the beginning material for cDNA libraries.

cDNA libraries give data about the articulation levels of mRNA. A genomic library will just offer us data on quality
portrayal as they happen in the chromosome.

An advantage of cDNA and cDNA libraries, which is another mark of detachment from genomic libraries, is that cDNA
doesn't have introns. This is very helpful when utilizing prokaryotic creatures for cloning since they don't have joining
capacities.

Colony Hybridization
This strategy distinguishes the DNA of premium utilizing a radiolabeled test that ties to the objective grouping.
Hybridization will be helpful when you know the arrangement of the quality you're keen on.

In this screening strategy, a nylon channel paper is utilized to recreate an expert plate containing settlements (every state
contains a homogenous populace of indistinguishable shut plasmid) by squeezing it onto the expert plate accordingly
moving cells from the provinces from the expert plate onto the nylon move paper. Then, at that point, radio named tests
including correlative oligos of the objective arrangement are added. The tests hybridize with DNA from the lysed cells.
Then, at that point, the channel paper is presented to X-beam which will once created, will permit perception of the
objective, and empower us to make an examination between the marked nylon paper and the expert plate to discover the
provinces containing our DNA of interest. The chose states are then picked and become on supplement medium.

Screening with nucleotide(oligo) probes

this technique will be helpful when you don't have the foggiest idea about the DNA arrangement. It is comparable from
multiple points of view to settlement hybridization; nonetheless, this method depends on the peptide grouping instead of the
DNA succession. From the peptide succession, a specialist can track down the conceivable DNA arrangement, produce a
reciprocal grouping and utilize that as a test/groundwork. From that point, the remainder of the procedure follows settlement
hybridization.

Immunological Screening
Here, nitrocellulose paper is laid on the petri dish where proteins will tie. The nitrocellulose is first brooded with the
essential immunizer, then, at that point with a radiolabeled auxiliary neutralizer. After advancement, the general area of the
state of interest can be resolved utilizing the uncovered X-beam film as an aide.

Subtractive Screening
The past techniques permit scientists to begin with some sort of known to screen their library. Subtractive screening, in any
case, is equipped for distinguishing novel quality articulation from mRNA. For instance, openness to another medication
treatment or natural pollutant is accepted to prompt special quality articulation from a control. Subtractive screening
(likewise called differential screening) analyzes this one of a kind articulation and eventually permits specialists to look at
the DNA being referred to against a control to discover a distinction in articulation.

C-DNA synthesis and library preparation

Synthesis of cDNA fake cDNA assumes a significant part in clinical exploration and analysis. Here we have clarified the
cycle of cDNA union.

mRNA isolation
The mRNA can be segregated utilizing the prepared to utilize mRNA disengagement unit. To detach mRNA from the
remainder of the RNA, oligo dT containing section is utilized in the segregation interaction.

When the mRNA is deciphered, the poly-A tail is added during the interaction called post-transcriptional alterations. What's
more, this separates the mRNA from the remainder of the single-abandoned RNAs.

The poly-A tail of the mRNA stays limited with the oligo dT containing section. After each round of washing, every one of
the RNAs are washed off just the mRNA stays in the segment.

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In the last advance, the mRNA is gathered in another cylinder by utilizing the elution support.

mRNA purification
Cleansed mRNA should need for the subsequent stage, for that, the mRNA is sanitized utilizing the refinement pack and the
oligo-dT correlative nucleotides are taken out from the poly-A tail by warming it tenderly. The refined mRNA is utilized for
the converse transcriptase PCR.

Selection of enzyme
A typical polymerase can't blend DNA from RNA. We need another kind of polymerase for that-an opposite transcriptase
polymerase.

An opposite transcriptase catalyst is a unique kind of polymerase segregatedfrom the retroviruses having the ability to
orchestrate cDNA from the mRNA.

The Avian Myeloblastomis infection turns around transcriptase and Moloney Murine Leukemia Virus invert transcriptase
are two financially accessible RTs utilized regularly in the cDNA library planning

Reverse transcripase PCR

Ordinary PCR is utilized for the combination of DNA from DNA while the converse transcriptase PCR is appropriate for
the blend of DNA from the RNA layout utilizing the opposite transcriptase catalyst.

DNA is combined back from the record or mRNA yet the means of the RT-PCR are practically like ordinary customary
PCR.

Formation of library
Presently we have the amplicons of a cDNA, the cDNA is currently embedded into the plasmid utilizing limitation
assimilation strategy.

Contingent on the size and sort of cDNA, various kinds of plasmids are utilized for developing diverse cDNA libraries. The
plasmid with the cDNA is currently embedded into the microscopic organisms and developed utilizing the supplement
media under aseptic conditions.

Normalizing the C-DNA

cDNA normalization diminishes the predominance of high wealth records and adjusts record fixations in a cDNA test,
subsequently significantly expanding the effectiveness of sequencing and uncommon quality revelation

In an eukaryotic cell, the mRNA populace comprises roughly 1% of absolute RNA with the quantity of records changing
from a few thousand to a many thousands.

Specific Nuclease Based TRIMMER Technology

Quick and dependable approach to balance bounty of various records in a cDNA populace and get ready full-length-
enhanced standardized cDNA pools/libraries for EST investigation and uncommon quality disclosu

1. Rapid and solid approach to eliminate rehashed records from cDNA library
2. Equalization of full-length-improved cDNA before library cloning
3. Simple technique, no actual partition steps
4. Fully viable with Illumina/Solexa, ABI/SOLiD and Roche/454 sequencing convention

The subsequent cDNA contains adjusted bounty of various records and can be utilized for development of cDNA libraries
and for direct sequencing, including high-throughput sequencing on the cutting edge sequencing stages (Roche/454,
ABI/SOLiD or Illumina/Solexa). The pack additionally incorporates extraordinary connectors permitting utilization of
Clontech SMART-based units for development of cDNA expected for Trimmer-2 standardization

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Results and Discussions:-

Identification of some genes
Screening of MlJAZ1 GENE(ML074)
The identification of MIJAZI gene was done with the help of MeJA treatment . The cDNA-AFLP tag was purged from the
gel, reamplified , and sequenced, uncovering an EST of 449 nucleotides that was intently homologous to the jasmonate
flagging repressor JAZ1/TIFY10A from Arabidopsis thaliana. The introductions for the PCR screening (forward and
opposite, 5′-TTTATTCCCCCAGCACTCTG-3′ and 5′-TCGGAGCTTGCCTTACTAGC-3′, individually) (Figure 1D) were
created on the cDNA-AFLP tag with the Primer3 program . Ensuing screening of the pool plasmid DNA uncovered that the
clone was available in pools 1, 6, 7, 8, and 12 . After settlement hybridization on the film of pool 7, three up-and-comer
provinces could be seen on the subsequent autoradiogram . Through colony PCR with the previously mentioned
preliminaries, two of the three up-and-comer settlements were found to contain the clone of interest . Sequencing of the two
positive clones showed that in the two clones an indistinguishable FL open understanding edge (ORF) of 819 nucleotides
happened, encoding a protein of 273 amino acids, from now on alluded to as MlJAZ1. The 5′ untranslated locale (UTR)
present in clone 1 was 153 nucleotides in length, though the 5′ UTR of clone 2 was just 36 nucleotides in length.
Investigation of the got MlJAZ1 succession showed that the protein contained the trademark tify and Jas spaces of the JAZ
protein family . The tify space and the C-terminal Jas area are described by the profoundly saved TIF[F/Y]XG and
SLX2FX2KRX2RX5PY amino corrosive successions, individually .

Identification of Maesa lanceolata genes

Some M. lanceolata cDNA-AFLP labels were chosen for additional investigation. PCR screening of the pool plasmid DNA
demonstrated that clones comparing to 52 of the 53 chose labels were available in no less than one of the 12 library
pools.Furthermore, clones relating to 23 labels were available in every one of the pools, proposing a high portrayal in the
library. Consequently, a first round of province hybridizations were performed, in which the 52 radioactively named cDNA-
AFLP labels were hybridized on one film of a pool for which the presence of a clone relating to the cDNA-AFLP tag was
affirmed by the PCR screening. For 45 labels, something like one competitor settlement was distinguished, however state
PCR uncovered that for 15 labels, all recognized applicant provinces were bogus positives. In this way, for 30 cDNA-AFLP
labels, affirmed states were acquired and accordingly sequenced, until a clone with a FL-coding arrangement was
recognized, bringing about the distinguishing proof of FL successions for 19 of the 53 at first chose cDNA-AFLP labels
(36%). For the leftover 33 cDNA-AFLP labels that happened in at least one of the pools, and for which no relating FL-
coding grouping was recognized, a second round of hybridizations was performed .until for each cDNA-AFLP label a
competitor state was distinguished, or until every one of the pools with a hit in the PCR screen were depleted. For all,
however one, of the 33 excess labels, competitor provinces were distinguished

Conclusions:-
cDNA-AFLP is a generally utilized, powerful, and reproducible instrument for genome-wide articulation investigation in
any organic entity, without the requirement for earlier succession information . The procedure is gotten from AFLP and
depends on the particular PCR enhancement of limitation sections from a twofold abandoned cDNA layout. To this end, the
twofold abandoned cDNA format is processed with limitation proteins, trailed by ligation of explicit connectors to the tacky
finishes of the processed cDNA. The intensified cDNA sections are envisioned on high-goal polyacrylamide gels, on which
the power of the parts mirrors the relative plenitude (duplicate number) of the comparing qualities across the examples . To
distinguish the differentially communicated qualities, the comparing cDNA-AFLP labels are decontaminated from the
polyacrylamide gel, reamplified, and sequenced. In any case, the subsequent cDNA-AFLP label groupings regularly don't
compare to FL-coding successions and, accordingly, don't give adequate arrangement data to utilitarian portrayal.

One of the usually utilized techniques to acquire a FL cDNA clone beginning from a halfway clone, for example, a cDNA-
AFLP tag or some other kind of EST, is the quick intensification of cDNA closes (RACE)- PCR procedure, in which
connectors ligated to the 3′ and 5′ finishes of the cDNA are utilized to specifically intensify these 3′ or 5′ cDNA parts by
PCR through a mix of quality explicit and connector explicit preliminaries . A typical issue related with RACE-PCRs is the
enhancement of vague PCR items because of the presence of one of the groundwork arrangements in all cDNAs . An option
in contrast to the RACE-PCR system is the screening of cDNA libraries. The conventional way of library screening is work
concentrated, particularly when evaluating for uncommon records . A more quick way to deal with acquire a clone of
interest from a cDNA library is by PCR screening of pooled clones. This methodology requires showing of individual
clones into microtiter wells, and is accordingly just viable for bountiful records . Then again, Self-Ligation of Inverse PCR
Products (SLIP) takes into consideration the synchronous evaluating for low bountiful records. In this method, the plasmid
of interest is enhanced from a little aliquot of the cDNA library through backwards PCR, utilizing preliminaries planned in

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inverse direction on the deficient cDNA. The got PCR parts are self-ligated, changed into Escherichia coli and plated.
Accordingly, singular settlements are picked for DNA confinement and sequencing . Nonetheless, like different RACE-PCR
approaches, this PCR-based library screening procedure is helpless to intensification curios. Subsequently, in view of the
tedious and work serious iterative screening of various cDNA libraries or the requirement for guided methodologies to deal
with singular clones in a RACE-PCR approach, to acquire the FL cDNA clone of enormous quantities of deficient cDNAs
in equal, may end up being a genuine test Applicant states were picked from the 24×24 cm Petri dishes and checked by
province PCR with the groundworks planned on the cDNA-AFLP tag. Positive clones were sequenced with the
groundworks planned on the pENTR222 vector of the Uncut Nanoquantity cDNA library, 5′-
ACGACGGCCAGTCTTAAGCTCGG-3′ and 5′-ACCATGTAATACGACTCACTATAGG-3′

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