Isolation of Exons from Cloned DNA by UNIT 6.

1
Exon Trapping
Exon trapping is an RNA polymerase chain reaction (PCR) method to clone expressed
sequences or exons directly from mammalian genomic DNA; Figures 6.1.1 and 6.1.4
outline two approaches. The basic steps in exon trapping are the insertion of fragments
of vertebrate DNA into one of several trapping vectors, transfection of recombinants into
mammalian cells, and selection of chimeric mRNA transcribed from vector promoters
using the technique of reverse transcription polymerase chain reaction (RT-PCR). The
resulting PCR products are then sequenced to identify the newly discovered exon and can
also be used to screen cDNA libraries to obtain a cDNA clone of the gene. It has proved
to be an important part of positional cloning strategies to identify human disease genes
such as that for Huntington disease. It can also be used to isolate exons from genomic
DNA without knowledge of the developmental time or place of gene expression.
The first basic protocol describes the method for trapping internal exons from cosmid
clones and the second basic protocol describes trapping of 3′ terminal exons. In 3′ terminal
exon trapping, subcloning of target DNA can be avoided by ligating it to the vector for
direct transfection (alternate protocol). A support protocol describes a rapid cloning
procedure using uracil DNA glycosylase.
CAUTION: Radioactive, biological, and chemical substances require special handling;
see APPENDIX 2A for guidelines.
NOTE: All reagents and equipment coming into contact with live cells must be sterile.
NOTE: Experiments involving PCR and RNA require extremely careful technique to
prevent contamination and RNA degradation; see APPENDIX 2D.

INTERNAL EXON TRAPPING FROM COSMID CLONES BASIC

PROTOCOL 1
Genomic DNA from cosmids is subcloned into the plasmid pSPL3. This subcloning
protocol is designed for cosmid clones—i.e., one cosmid clone is digested and shotgun
subcloned into pSPL3. The procedure can be adapted for genomic DNA cloned in
plasmids, phage, or yeast artificial chromosomes (YACs). For example, transfections may
contain a single subclone or complex pools of subclones comprising as many as ten
cosmid equivalents of DNA (∼400 kb). Subcloning is followed by transfection of
subcloned DNA into COS-7 cells. After transient expression, RNA is harvested and
reverse transcribed using a vector-specific oligonucleotide to yield first-strand cDNA.
After digestion of the RNA template, an initial round of PCR is performed, followed by
digestion with BstXI to remove PCR products that do not contain exons. A second round
of PCR is performed, followed by rapid cloning into a phagemid vector using uracil DNA
glycosylase (support protocol).

Identifying
Candidate Genes
in Genomic DNA

Contributed by Paul E. Nisson, Paul C. Watkins, and David B. Krizman 6.1.1

Current Protocols in Human Genetics (1994) 6.1.1-6.1.27
Copyright © 2000 by John Wiley & Sons, Inc. Supplement 3
 B B B BB B B B B
cosmid
DNA

BamHI SA SD BamHI

+
SD SA
BstXI MCS BstXI
half-site half-site
BstXI SstI NotI BamHI BstXI
pSPL3
EcoRI XhoI XmaIII PstI EcoRV

cryptic splice-donor

SD BamHI SA SD BamHI NdeI SA

subcloned
MCS MCS DNA

RNA after
SD6 SA2 splicing
dUSD2 dUSA4

dUSD2 dUSA4 trapped exon

after PCR

Figure 6.1.1 Schematic representation of internal exon trapping. Cosmid DNA is digested at
specific restriction sites with a restriction enzyme (e.g., BamHI). In this example, a single exon (gray
box) is contained in a BamHI fragment. The exon is flanked by splice-acceptor (SA) and splice-donor
(SD) sites. pSPL3 vector is prepared for subcloning at the same restriction site. The multiple cloning
site (MCS) of pSPL3 contains several restriction sites. EcoRI and EcoRV sites are contained within
BstXI sites. The black boxes represent HIV tat and rabbit β-globin exon sequences in the pSPL3
vector. The splice-donor and splice-acceptor sites of pSPL3 contain BstXI half-sites. These sites
form a complete BstXI site after splicing events that fail to capture a “trapped” exon and can therefore
be used to eliminate vector background in the subsequent PCR amplification. After subcloning of
genomic DNA into pSPL3, DNA is isolated and transfected into COS-7 cells. Cytoplasmic RNA is
isolated for RNA-based PCR analysis. After generation of cDNA, a first round of PCR is performed
using the outside primer pair SD6 and SA2. A second round of PCR is performed using the nested
Isolation of Exons primers dUSD2 and dUSA4 to provide specificity and to install DNA sequences that allow uracil
from Cloned DNA glycosylase (UDG) cloning of the PCR products. Trapped exons are recognized after gel
DNA by Exon
Trapping analysis of the PCR products. Occasional but infrequent use of the cryptic splice-donor site can be
detected by digestion with NdeI.
6.1.2
Supplement 3 Current Protocols in Human Genetics
Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock
solutions, see APPENDIX 2; for suppliers, see SUPPLIERS APPENDIX.
Cosmid clones
1 µg pSPL3 vector (Fig. 6.1.2; GIBCO/BRL)
Restriction endonucleases: BstXI and one or more of the following for
subcloning cosmids into pSPL3: BamHI, BglII, SstI, NotI, XhoI, XmaIII,
PstI, and 10× buffers
7.5 M ammonium acetate
100% and 70% ethanol
TE buffer, pH 8.0
Calf intestine alkaline phosphatase (CIP)
1 U/µl T4 DNA ligase (in Weiss units)
5× T4 DNA ligase buffer (APPENDIX 2; make 5× and substitute 25% PEG
8000 for BSA)
Transformation-competent E. coli cells (CPMB UNIT 1.8)
LB medium and plates containing 100 µg/ml ampicillin (LB/ampicillin; APPENDIX 2)
COS-7 cells (ATCC #CRL-1651)
Complete DMEM/10%, 20%, and 0% FBS (37°C, APPENDIX 3G)
Cationic lipid (e.g., LipofectACE; GIBCO/BRL)
Diethylpyrocarbonate (DEPC)-treated H2O (UNIT 7.1)
20 µM oligonucleotide SA2: 5′-ATCTCAGTGGTATTTGTGAGC-3′
5× first-strand buffer (see recipe)
0.1 M dithiothreitol (DTT)
10 mM 4dNTP mix (APPENDIX 2) in DEPC-treated H2O
200 U/µl reverse transcriptase
2 U/µl RNase H
5 U/µl Taq DNA polymerase
10× Taq DNA polymerase buffer: 500 mM KCl/100 mM Tris⋅Cl, pH 8.3
(store ≤18 months at −20°C)
50 mM MgCl2
10 mM 4dNTP mix in sterile H2O (APPENDIX 2)
20 µM oligonucleotide SD6: 5′-TCTGAGTCACCTGGACAACC-3′
Mineral oil
20 µM oligonucleotide dUSA4:
5′-CUACUACUACUACACCTGAGGAGTGAATTGGTCG-3′
20 µM oligonucleotide dUSD2:
5′-CUACUACUACUAGTGAACTGCACTGTGACAAGCTGC-3′
2% agarose gel
6-well tissue culture plates with 3.5-cm wells
1.5-ml polystyrene microcentrifuge tube, sterile
0.5-ml polypropylene microcentrifuge tubes
Thermal cycler
42° and 55°C water baths
Additional reagents and equipment for phenol extraction and ethanol precipitation
of DNA (APPENDIX 3C), E. coli transformation (CPMB UNIT 1.8), alkaline lysis
miniprep of plasmid DNA (UNIT 5.3 & CPMB UNIT 1.6), mammalian cell culture
(APPENDIX 3G), liposome-mediated transfection (CPMB UNIT 9.4) or transfection by
electroporation (CPMB UNIT 9.3), total RNA isolation by the rapid guanidinium
method (UNIT 10.4 & CPMB UNIT 4.2), RNA quantitation (APPENDIX 3D), and agarose
gel electrophoresis (UNIT 2.7) Identifying
Candidate Genes
in Genomic DNA

6.1.3
Current Protocols in Human Genetics Supplement 3
 SD H IV t at int ron SA
MCS

exon exon

pA
SV40

pSPL3
6031 bp

ori

Ap r

Figure 6.1.2 pSPL3 vector. The vector contains sequences that allow replication in E. coli and
COS-7 cell hosts (bacterial and SV40 origins of replication are both present). An ampicillin-resis-
tance marker (Apr) allows for selection of subclones. A multiple cloning site (MCS) interrupts the
HIV tat intron and provides several restriction sites for subcloning genomic DNA. After transfection
of COS-7 cells, replication and transcription occur at high levels facilitated by the SV40 origin of
replication and promoter (SV40), respectively. Processing of the transcript results in removal of the
HIV tat intron via splicing in which vector exons that contain sequences from HIV and rabbit
beta-globin are combined at the splice-donor (SD) and splice-acceptor (SA) sites. Cytoplasmic RNA
is polyadenylated [pA = SV40 poly(A) addition recognition sequence]. Exons are “trapped” from
genomic DNA cloned into pSPL3 as a result of interaction of the vector splice sites (derived from
HIV tat) with splice sites flanking exons contained in genomic DNA.

NOTE: An Exon Trapping System kit which contains plasmids and many of the reagents
and solutions necessary for this protocol is available from GIBCO/BRL.
NOTE: All tissue culture incubations are performed in a humidified 37°C, 5% CO2
incubator unless otherwise specified.

Prepare cosmid and pSPL3 for subcloning (day 1)

1. Digest ≥1 µg of cloned genomic (cosmid) DNA and 1 µg of pSPL3 DNA in separate
1.5-ml microcentrifuge tubes with appropriate restriction endonuclease(s).
The multiple cloning site (MCS) of pSPL3 contains unique restriction endonuclease sites
for EcoRI, SstI, XhoI, NotI, XmaIII, PstI, BamHI, and EcoRV, permitting a great deal of
flexibility in experimental design. For routine analysis of cosmids, a combination of BamHI
and BglII single and double digests of cosmid DNA cloned into the BamHI site of pSPL3
generally yields good results.
2. Phenol extract cosmid and pSPL3 DNAs. Ethanol precipitate DNAs using 1⁄2 vol of
7.5 M ammonium acetate and 2 vol of 100% ethanol. Wash the pellet with 70%
ethanol.
3. Dissolve DNA pellets in TE buffer, pH 8.0, to give 250 µg/ml final concentration.
4. Dephosphorylate linearized pSPL3 DNA with CIP, following procedure rec-
Isolation of Exons comended by supplier.
from Cloned
DNA by Exon Alternatively, two different restriction enzymes can be used to prepare pSPL3 for sub-
Trapping cloning. In this case, dephosphorylation is not required.

6.1.4
Supplement 3 Current Protocols in Human Genetics
Shotgun subclone genomic DNA into pSPL3 (days 1 to 3)
5. Add the following to a microcentrifuge tube:
0.25 µg pSPL3 DNA (from step 4)
0.25 to 1 µg genomic (cosmid) DNA (from step 3)
2 µl 5× T4 DNA ligase buffer
1 U T4 DNA ligase
H2O to 10 µl final.
Mix gently and incubate 1 hr at room temperature.
Include a control reaction, containing all the above components without genomic DNA, to
assess the level of vector self-ligation.
BstXI digestion is used to detect trapped exons juxtapositioned with the vector cryptic
splice-donor site of the pSPL3 plasmid. Subcloning into either EcoRI or EcoRV sites will
inactivate the overlapping BstXI sites and make it necessary to digest putative exon-con-
taining clones with NdeI to determine if the vector cryptic splice-donor site has been used
(Fig. 6.1.2; see Critical Parameters for a more detailed explanation).
6. Place sample on ice. Transform E. coli with ligated DNA using either chemical or
electrocompetent cells.
7. Inoculate 5 ml LB/ampicillin medium with 0.5 ml cells from each transformation and
incubate overnight at 37°C in a standard room-air incubator.
8. Plate 0.1 and 0.01 ml of each transformation on LB/ampicillin plates to assess the
degree of vector religation. Incubate overnight at 37°C in a standard room-air
incubator. Compare the numbers of colonies on the two plates.
Expect 10 to 100 colonies on the 0.01-ml experimental plate. If >10% of recombinants are
vector religations, dephosphorylated vector should be prepared again and the transforma-
tion should be repeated.
9. Isolate DNA from the 5 ml culture (step 7) by the alkaline lysis miniprep procedure.
Efficiency of shotgun subcloning can be assessed by digesting the DNA with the same
restriction enzyme used for subcloning and comparing the sizes of the resulting fragments
to those of the digested source (cosmid) DNA.

Carry out lipid-mediated transfection of COS-7 cells (days 4 and 5)

10. Sixteen hours before starting the transfection, plate COS-7 cells in 37°C supple-
mented DMEM/10% FBS at the appropriate density on 6-well tissue culture plates
(one well per transfection).
Each well should be ∼75% to 80% confluent (∼5 × 105 cells).
COS-7 cells, a commonly used tissue culture cell line, have a doubling time of ∼20 hr. Cells
obtained from ATCC are frozen and should be passaged at least once to determine that
they are growing with the proper doubling time. It is recommended that a 70-cm2 flask of
COS-7 cells be maintained, passaging when the cells reach 100% confluence (once or twice
a week) by trypsinizing the cells and diluting them 1:50 in fresh medium.
11. For each transfection, add an optimized amount of cationic lipid to 100 µl serum-free
medium in a sterile polystyrene tube, mix gently, and incubate 5 min at room
temperature.
Polystyrene tubes should be used for dilutions of the lipid reagent because it has a tendency
to stick to polypropylene.
Optimal amount of cationic lipid and DNA, as well as appropriate medium and desired
Identifying
level of confluence of COS-7 cells, should be determined according to the supplier’s Candidate Genes
recommendations. in Genomic DNA

6.1.5
Current Protocols in Human Genetics Supplement 3
 Although electroporation can be used to introduce genomic DNA subcloned in pSPL3 into
COS-7 cells, transfection with cationic lipids—also known as liposome-mediated transfec-
tion—has been found to be comparable in efficiency and less costly in time and materials.
12. For each transfection, place 1 µg DNA (purified from subclones; step 9) into 100 µl
supplemented DMEM (serum-free) in a separate sterile polystyrene tube.
13. Combine cationic lipid and DNA solutions, mix gently, and incubate 15 min at room
temperature to form DNA/cationic lipid complexes.
14. Aspirate medium from COS-7 cell cultures. Add 2 ml supplemented 37°C DMEM
(serum-free) to plates and incubate 5 min at 37°C.
15. Add 0.8 ml supplemented 37°C DMEM (serum-free) to DNA/cationic lipid complex
(from step 13).
16. Remove medium from culture plate.
This wash serves to remove any trace of serum that may lower transfection efficiency.
17. Add one tube of DNA/cationic lipid complex (from step 15) to plate and incubate 5
to 6 hr.
18. Add 1 ml supplemented 37°C DMEM/20% FBS and incubate 24 hr.
Plates will be nearly confluent after 24 hr.
19. Remove cells from tissue culture dish by trypsinization. Prepare total RNA by rapid
guanidinium method and dissolve in 50 µl DEPC-treated water. Measure RNA
concentration.
Appropriate precautions should be taken when working with RNA. All water and salt
solutions, except those containing Tris, should be DEPC-treated. All glass and plasticware
must be RNase-free (CPMB UNIT 4.1).
Although other methods for isolating total RNA can be used, the acidic phenol–guanidinium
thiocyanate reagent developed by Chomczynski and Sacchi (1987; UNIT 10.4 & CPMB UNIT 4.2)
is recommended because of its ease of use and the reproducible quality and quantity of RNA
obtained. Normally, 50 to 100 ìg of total RNA are obtained per 3.5-cm plate.

Synthesize first-strand cDNA (day 6)

20. Add the following to a 0.5-ml polypropylene microcentrifuge tube:
1 µl 20 µM oligonucleotide SA2
1 to 3 µg RNA
DEPC-treated H2O to 12.0 µl final.
Incubate mixture 5 min at 70°C, then incubate 1 min on ice.
21. Microcentrifuge briefly at high speed and add the following at room temperature:
4 µl 5× first-strand buffer
2 µl 0.1 M DTT
1 µl 10 mM 4dNTP mix in DEPC-treated H2O.
Load the tube into the thermal cycler and initiate a program that will run 10 min at
70°C, 30 min at 42°C, and 15 min at 55°C.
It is convenient to hold the thermal cycler at 70°C while preparing the reaction mix.

Isolation of Exons
from Cloned
DNA by Exon
Trapping

6.1.6
Supplement 3 Current Protocols in Human Genetics
22. Mix gently, microcentrifuge briefly at high speed, and incubate 2 min at 42°C.
23. Add 200 U reverse transcriptase and mix gently. Incubate 30 min at 42°C, then
incubate 5 min at ∼55°C.
24. Add 1 µl RNase H, mix gently, and incubate 10 min at 55°C.
RNase H digestion is conducted at a temperature at which reverse transcriptase is inactive.
This reduces the possible synthesis of second-strand products from hairpin structures
(molecules that are partially double-stranded and provides a priming site for reverse
transcriptase).
25. Microcentrifuge briefly at high speed and place on ice. Remove 8 µl for primary PCR
amplification.
The experiment may be stopped at this point by storing the tube at −20°C.

Perform primary PCR (day 6)

26. Add the following to a 0.5-ml polypropylene microcentrifuge tube:
5 µl reverse transcriptase reaction mixture (from step 25)
5 µl 10× Taq DNA polymerase buffer
1.5 µl 50 mM MgCl2
1 µl 10 mM 4dNTP mix in sterile H2O
2.5 µl 20 µM oligonucleotide SA2
2.5 µl 20 µM oligonucleotide SD6
Sterile H2O to 47.5 µl final.
Mix and overlay with a drop of mineral oil.
27. Preheat thermal cycler to 94°C. Place the reaction tube in the thermal cycler and
incubate 5 min at 94°C.
28. Reduce thermal cycler temperature and hold tubes at 80°C. Add 2.5 U Taq DNA
polymerase diluted to a final volume of 2.5 µl in 1× Taq DNA polymerase buffer.
29. Carry out PCR using the following amplification cycles:
6 cycles: 1 min 94°C (denaturation)
1 min 60°C (annealing)
5 min 72°C (extension)
1 cycle: 10 min 72°C (extension)
Final step: indefinitely 55°C (hold).
First-strand cDNA is PCR amplified using vector-directed oligonucleotide SA2 and SD6.
30. Add 25 U BstXI and incubate overnight at 55°C.
Double-stranded PCR products are digested with BstXI to eliminate two classes of
undesirable products: those that result from pSPL3-derived molecules containing only
vector sequences, and those that have used the cryptic splice-donor site (Fig. 6.1.1; see
Critical Parameters).
31. Add 5 U BstXI and incubate for an additional 2 hr at 55°C.

Identifying
Candidate Genes
in Genomic DNA

6.1.7
Current Protocols in Human Genetics Supplement 3
 Perform secondary PCR (day 7)
32. Add the following to a 0.5-ml polypropylene microcentrifuge tube:
5 µl BstXI-treated primary PCR product
4.5 µl 10× Taq DNA polymerase buffer
1.5 µl 50 mM MgCl2
1 µl 10 mM 4dNTP mix in distilled H2O
1 µl 20 µM oligonucleotide dUSA4
1 µl 20 µM oligonucleotide dUSD2
Sterile H2O to 47.5 µl final.
Mix and overlay with a drop of mineral oil.
Secondary PCR primers dUSA4 and dUSD2 contain an additional 12 nucleotides at the
5′ end. These primers were designed specifically to enable cloning of the PCR products
using uracil DNA glycosylase (support protocol) but could be modified to allow cloning of
the PCR products by other methods.
33. Heat thermal cycler to 94°C. Place reaction tube into thermal cycler and incubate 5
min at 94°C.
34. Reduce thermal cycler temperature and hold tubes at 80°C. Add 2.5 U Taq DNA
polymerase diluted to a final volume of 2.5 µl in 1× Taq DNA polymerase buffer.
35. Carry out PCR using the following cycles:
30 cycles: 1 min 94°C (denaturation)
1 min 60°C (annealing)
3 min 72°C (extension)
1 cycle: 10 min 72°C (extension)
Final step: indefinitely 4°C (hold).
36. Electrophorese 4 µl of PCR reaction products on a 2% agarose gel and identify
reactions that contain PCR products.
These PCR products can be used for cloning with uracil DNA glycosylase (support
protocol). They may be stored at −20°C.

BASIC 3′ TERMINAL EXON TRAPPING TO IDENTIFY GENES IN

PROTOCOL 2 CLONED DNA
3′ terminal exon trapping involves ligation of an incomplete transcription unit (vector
pTAG4, Fig. 6.1.3), consisting of a strong SV40 promoter and two internal exons from
the human adenovirus, to genomic target DNA from plasmid, phage, cosmid, P1, or YAC
clones. If the target DNA contains a 3′ terminal exon, the truncated transcription unit from
the trapping vector will be completed and a stable polyadenylated mRNA will be made
from the chimeric precursor RNA. A cDNA can then be reverse transcribed from the
mRNA and amplified by rapid amplification of cDNA ends (3′ RACE), which uses the
reverse transcription polymerase chain reaction (RT-PCR). RT-PCR amplification prod-
ucts are then used for further mapping and sequencing (Fig. 6.1.4).
Target DNA and the trapping vector pTAG4 are digested with the same restriction
endonuclease and shotgun subcloned. Recombinants are selected in Escherichia coli, and
a mixture of all recombinants is transfected into COS-7 cells for expression of vector-de-
Isolation of Exons rived mRNA. This is followed by RT-PCR to obtain copies of the newly discovered 3′
from Cloned
DNA by Exon terminal exon(s). Individual PCR products can be cloned using the uracil DNA glycosy-
Trapping lase (UDG) approach (support protocol; see Fig. 6.1.6) for further analysis.
6.1.8
Supplement 3 Current Protocols in Human Genetics
 multiple
5′ EcoRIBamHI cloning
3′ site
2 Bgl II
BssHII
5′ NheI
NotI/EagI
3′ 1 PstI
NarI
MluI
SV40 SplI
early NruI
region ColE1
pTAG4 ori
~4000 bp

f1

Ap r

Figure 6.1.3 pTAG4 vector. pTAG4 is a pUC-based vector containing the SV40 promoter/en-
hancer early region, which drives transcription when inserted into COS-7 cells that express the
large T antigen from SV40 virus. Leader exons 1 and 2 (1, 2), with the natural intron, are derived
from human adenovirus 2. These are downstream of the promoter, forming an incomplete transcrip-
tion unit that lacks a 3′ terminal exon to direct polyadenylation of transcripts from the vector. Arrows
indicate direction of transcription. The arrowhead indicates the alternate 5′ splice site within the
multiple cloning site.

Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock
solutions, see APPENDIX 2; for suppliers, see SUPPLIERS APPENDIX.
pTAG4 vector (Fig. 6.1.3; GIBCO/BRL), purified by CsCl gradient (UNIT 5.3 &
CPMB UNIT 1.7)
Restriction endonuclease that cuts within pTAG4 multiple cloning site (MCS):
EcoRI, BamHI, BglII, BssHII, NheI, EagI, NotI, PstI, NarI, MluI, or SplI,
and appropriate 10× buffer
Calf intestine alkaline phosphatase (CIP)
TE buffer, pH 8.0
Genomic target DNA: plasmid, cosmid, phage, P1, or YAC clones
β-agarase
4 M NaCl
0.8% agarose gel
5× T4 DNA ligase buffer (in Weiss units)
1 U/ml T4 DNA ligase
Transformation-competent E. coli cells (CPMB UNIT 1.8)
LB medium and plates containing 100 µg/ml ampicillin (LB/ampicillin; APPENDIX 2)
COS-7 cells (ATCC #CRL-1651)
Complete DMEM/20%, 10%, and 0% FBS (APPENDIX 3G)
Cationic lipid (e.g., LipofectACE, GIBCO/BRL)
Diethylpyrocarbonate (DEPC)-treated H2O (UNIT 7.1) Identifying
Candidate Genes
10× DNase I buffer (see recipe) in Genomic DNA

6.1.9
Current Protocols in Human Genetics Supplement 3
 genomic DNA fragment
3′ terminal exon
3′ (627 bp average)
*

5′ 3′ 5′
1 2
SV40 early promoter
transfect into COS-7 cells
(basic protocol 1, steps 10-18)

isolate RNA after 16 hr

(basic protocol 2, steps 10-12)
1 2 * AAAAAAAAAn mRNA
TTTTTTT
X
AP primer
carry out RT-PCR
(basic protocol 2, steps 13-17)
1 2 UAP
* TTTTTTT X cDNA
Ad2
carry out PCR
(basic protocol 2, steps 18-25)

* AAAAAAAAA X amplified product

2 TTTTTTTTT X containing 3′ terminal exon

Figure 6.1.4 Schematic representation of 3′ terminal exon trapping. Restriction fragments of

genomic DNA are inserted into pTAG4 multiple cloning site. Recombinant vector is transfected into
COS-7 cells to induce transcription from SV40 promoter (arrow indicates direction of transcription).
Transient RNA is harvested after 16 hr and reverse transcribed with AP primer, which relies on
presence of poly(A) tail to induce reverse transcription. Resulting single-stranded cDNA is then
amplified with primers specific for vector-derived viral sequences and the X sequence (an engi-
neered sequence that will not base-pair with any endogenous COS-7 mRNAs) downstream of the
poly(T) region of the AP primer. Trapped exons are recognized after gel analysis of PCR products
and subsequent sequencing. Use of UAP and AP primers for RT-PCR is the basis for 3′ RACE
technology. If original genomic DNA fragment contained a 3′ terminal exon with a poly(A) signal (*),
the transcription unit from pTAG4 will be completed and a mature, selectable mRNA made in COS-7
cells.

1 U/µl DNase I
5 ng/µl oligonucleotide AP (adapter primer):
5′-AAGGATCCGTCGACATCGATAATACGAC(T)17-3′
20 mM EDTA, pH 8.0
H2O, sterile
Mineral oil
5× first-strand buffer (see recipe)
0.1 M dithiothreitol (DTT)
25 mM 4dNTP mix in DEPC-treated H2O (APPENDIX 2)
200 U/µl Superscript II reverse transcriptase (GIBCO/BRL)
Isolation of Exons 2.6 U/µl RNase H
from Cloned
DNA by Exon 10× Taq DNA polymerase buffer: 500 mM KCl/100 mM Tris⋅Cl, pH 8.3
Trapping (store <18 months at −20°C)
6.1.10
Supplement 3 Current Protocols in Human Genetics
 25 mM MgCl2
50 µM oligonucleotide SV40P:
5′-AGCTATTCCAGAAGTAGTGA-3′
50 µM oligonucleotide UAP:
5′-CUACUACUACUAGTCGACATCGATAATACGAC-3′
5 U/µl Taq DNA polymerase
50 µM oligonucleotide Ad2:
5′-CAUCAUCAUCAUCAGTACTCTTGGATCGGA-3′
6-well tissue culture plates with 3.5-cm wells
1.5-ml polystyrene microcentrifuge tubes, sterile
42°, 55°, and 70°C water baths
0.5-ml polypropylene microcentrifuge tubes
Thermal cycler
Additional reagents and equipment for agarose gel electrophoresis (UNIT 2.7),
purification of DNA using glass beads (CPMB UNIT 2.1), alkaline lysis preparation
of plasmid, cosmid, or P1 DNA (UNIT 5.3 & CPMB UNIT 1.6) preparation of phage
DNA (CPMB UNIT 1.13), column purification of DNA (CPMB UNIT 2.14), phenol
extraction and ethanol precipitation of DNA (APPENDIX 3C), purification of YAC
DNA by PFGE (UNIT 5.7), total RNA isolation by the rapid guanidinium method
(UNIT 10.4 & CPMB UNIT 4.2), and RNA quantitation (APPENDIX 3D)
NOTE: A kit containing pTAG4 and all the reagents and materials necessary to perform
3′ terminal exon trapping can be obtained from GIBCO/BRL.
NOTE: Tissue culture incubations of COS-7 cells should be carried out in a humidified,
5% CO2, 37°C incubator.

Prepare trapping vector pTAG4

1. Digest to completion ≥20 µg purified pTAG4 vector DNA with a restriction endonu-
clease that cuts within the pTAG4 multiple cloning site (Fig. 6.1.3).
The restriction endonuclease should be the same as the one used to digest target DNA in
step 6a or 7b.
EcoRI, which is very reliable and readily available, has been extensively utilized as the
enzyme of choice for digesting pTAG4 and target DNA.
2. Dephosphorylate linearized pTAG4 with CIP, following the procedure recommended
by the supplier.
3. Electrophorese dephosphorylated pTAG4 on a 0.8% agarose gel. Cut out the band at
4.0 kb corresponding to linearized vector, and purify DNA using glass beads.
Commercial kits (e.g., GlassMAX, GIBCO/BRL; Glas-Pac, National Scientific Supply;
GeneClean, Bio101; and Qiaex Gel Extraction Kit, Qiagen) are available for purification
of DNA by the glass-bead method. Other techniques for purifying DNA from agarose gel
slices can be used in place of the glass-bead method (see CPMB UNIT 2.7).
4. Resuspend purified vector DNA to a final concentration of 500 ng/µl in TE buffer,
pH 8.0, and store at 4°C until ready to use.

Prepare target DNA

For genomic DNA cloned in plasmids, cosmids, phage, and P1:
5a. Prepare target DNA by standard methods (e.g., alkaline lysis).
Identifying
Clones propagated in E. coli (plasmid, cosmid, P1, and phage) are relatively free of Candidate Genes
contaminating chromosomal DNA and proteins after standard DNA preparation. CsCl in Genomic DNA

6.1.11
Current Protocols in Human Genetics Supplement 3
 purification is not necessary as DNA column purification (CPMB UNIT 2.14) gives adequate
results.
6a. Digest ≥5 µg target DNA with same restriction endonuclease as used for digestion
of pTAG4 in step 1.
7a. Phenol extract and ethanol precipitate restriction enzyme–digested target DNA.
8a. Resuspend target DNA in TE buffer to a final concentration of 500 ng/µl. Analyze an
aliquot on a 0.8% agarose gel. Store remaining DNA solution at 4°C until ready to
proceed. Continue with step 9.
For YAC clone DNA:
5b. Purify ≥2 µg YAC DNA by pulsed-field gel electrophoresis. Digest agarose by
treating gel slice with β-agarase according to manufacturer’s instructions. Add 4 M
NaCl to 0.5 M final concentration and mix gently. Place on ice 15 min, then
microcentrifuge 15 min at maximum speed, 4°C.
If yield of YAC DNA is poor, ≥1 ìg may be used.
6b. Carefully transfer supernatant into new microcentrifuge tubes, adding 500 µl to each
tube. Ethanol precipitate DNA in each tube, incubating 1 hr at −20°C.
7b. Resuspend precipitated YAC DNA in each tube in 10 µl TE buffer and pool tubes.
Digest YAC DNA with same restriction endonuclease used to digest pTAG4.
8b. Phenol extract and ethanol precipitate restriction enzyme–digested YAC DNA. Re-
suspend pellet in 5 µl TE buffer and analyze an aliquot on a 0.8% agarose gel. Store
remaining DNA solution at 4°C until ready to proceed. Continue with step 9.
Purification of YAC DNA by this method shears the DNA to roughly 50 kb; however, the
DNA is still large enough for restriction enzyme digestion and is suitable for exon trapping.

Subclone DNA and transfect COS-7 cells

9. Follow steps 5 to 18 of the first basic protocol to shotgun subclone the genomic DNA
and to transfect COS-7 cells, except use purified pTAG4 vector DNA (step 4) in place
of the pSPL3 DNA and restriction enzyme–digested target DNA (step 8a or b) in
place of cosmid DNA.
10. Remove transfected COS-7 cells from plate by trypsinization and prepare total RNA
by rapid guanidinium method. Dissolve RNA pellet in 17 µl DEPC-treated water.
Many kits for isolating total RNA by this method are available from biotechnology supply
companies.
Appropriate precautions should be taken when working with RNA. All water should be
sterile and DEPC-treated. Use of glassware should be avoided. Polypropylene microcen-
trifuge tubes and sterilized pipet tips should be used because they are sufficiently free of
RNases. Gloves should always be worn because hands are the main source of RNases.
11. Set up the following reaction (20 µl total):
17 µl total RNA (from step 10)
2 µl 10× DNase I buffer
1 µl 1 U/µl DNase I.
Incubate 15 min at room temperature.

Isolation of Exons DNase I treatment of RNA is important before performing RT-PCR; it removes DNA that
from Cloned might be used as a template for reverse transcriptase.
DNA by Exon
Trapping

6.1.12
Supplement 3 Current Protocols in Human Genetics
12. Phenol extract and ethanol precipitate RNA, using same procedure as for DNA.
Resuspend RNA in 20 µl DEPC-treated water and measure RNA concentration.

Synthesize cDNA
13. Set up reverse transcription reaction in a 0.5-ml polypropylene microcentrifuge tube
as follows:
1 µl 5 ng/µl oligonucleotide AP
5 µg total RNA (from step 12)
1 µl 20 mM EDTA
Sterile H2O to 20 µl.
Oligonucleotide AP (adaptor primer) is 45 nucleotides in length and consists of (T)17 at
the 3′ end that will prime cDNA from the poly(A) tail of all mRNA. The remainder of the
AP primer consists of an engineered sequence that will not base-pair with any endogenous
sequences from COS-7 mRNA species (the X sequence; Fig. 6.1.4). The resulting cDNA can
be amplified with 5′ primers specific for vector sequences and 3′ primers specific for the
engineered tail sequence of oligonucleotide AP. This is the 3′ RACE technique illustrated
in Figure 6.1.4.
14. Incubate 3 min in a 70°C water bath. Transfer to 42°C water bath and incubate 5 min.
15. Prepare the following mixture (30 µl total):
12 µl H2O
10 µl 5× first-strand buffer
5 µl 0.1 M DTT
1 µl 25 mM 4dNTP mix
2 µl 200 U/µl Superscript II reverse transcriptase.
Preheat to 42°C and add to the reaction tube from step 14 (50 µl final volume).
Incubate 30 min at 42°C.
16. Heat reaction 5 min at 55°C, then add 1 µl of 2.6 U/µl RNase H. Incubate an additional
10 min at 55°C.
17. Ethanol precipitate cDNA and resuspend in 50 µl TE buffer.
Resuspended cDNA may be stored at −20°C for several months, until ready for amplifica-
tion.

Perform primary PCR

18. Set up primary PCR mix as follows (97 µl total):
74 µl sterile H2O
5 µl cDNA
10 µl 10× Taq DNA polymerase buffer
5 µl 25 mM MgCl2
1 µl 25 µM 4dNTP mix
1 µl 50 µM oligonucleotide SV40P
1 µl 50 µM oligonucleotide UAP.
Mix gently and overlay mixture with 100 µl mineral oil.
Oligonucleotide SV40P is specific for the portion of the cDNA derived from the SV40
promoter, and oligonucleotide UAP (universal amplification primer) is specific for the tail
region of the AP primer.
19. Mix 0.5 µl of 5 U/µl Taq DNA polymerase with 2.5 µl sterile water and store on ice. Identifying
Candidate Genes
in Genomic DNA

6.1.13
Current Protocols in Human Genetics Supplement 3
 20. Incubate PCR reaction 3 min at 94°C. Reduce thermal cycler temperature to 80°C
and add 3 µl diluted Taq DNA polymerase.
Hot-Start PCR, the addition of Taq DNA polymerase after the temperature has been raised
above the denaturing temperature (94°C), lowers the amount of nonspecific priming and
polymerization that is induced when oligonucleotides anneal to cDNA before optimal
annealing temperature has been reached.
21. Carry out PCR using the following amplification cycles:
20 cycles: 30 sec 94°C (denaturation)
30 sec 55°C (annealing)
2 min 72°C (extension)
1 cycle: 5 min 72°C (extension)
Final step: indefinitely 4°C (hold).

Perform secondary PCR

22. Set up secondary PCR mix as follows (97 µl total):
1 µl 1/10 dilution of primary PCR product (step 21)
78 µl H2O
10 µl 10× Taq DNA polymerase buffer
5 µl 25 mM MgCl2
1 µl 25 µM 4dNTP mix
1 µl 50 µM oligonucleotide Ad2
1 µl 50 µM oligonucleotide UAP.
Primers Ad2 and UAP have been engineered to contain UDG-clonable ends. PCR products
generated from 3′ terminal exon trapping can be efficiently cloned utilizing UDG cloning
technology (support protocol; Fig. 6.1.6).
Oligonucleotide Ad2 is specific for the second exon from the adenovirus sequence within
pTAG4. Use of secondary PCR in conjunction with the Ad2 and UAP primers generates a
hemi-nested PCR reaction that imparts a great deal of specificity to RT-PCR.
23. Dilute Taq DNA polymerase and initiate hot-start PCR (steps 19 and 20), adding 3
µl diluted Taq DNA polymerase to the secondary PCR mix.
24. Carry out PCR using the following amplification cycles:
30 cycles: 30 sec 94°C (denaturation)
30 sec 55°C (annealing)
2 min 72°C (extension)
1 cycle: 5 min 72°C (extension)
Final step: indefinitely 4°C (hold).
25. Electrophorese 10 µl PCR solution in a 1.2% agarose gel and identify reactions that
contain PCR products.
Individual PCR products can be isolated from a gel and subcloned using UDG cloning
(support protocol). Alternatively, a library of all the PCR products of a single reaction can
be made using the UDG cloning approach.
Subclones containing putative 3′ terminal exons should be sequenced for confirmation.

Isolation of Exons
from Cloned
DNA by Exon
Trapping

6.1.14
Supplement 3 Current Protocols in Human Genetics
3′ TERMINAL EXON TRAPPING USING DIRECT ALTERNATE
LIGATION/TRANSFECTION PROTOCOL
This protocol describes direct ligation/transfection for presentation of target DNA to
pTAG4 (Fig. 6.1.5). By double-digesting the vector with both the enzyme used to cut the
target DNA and NruI, a blunt cutter, ligated target and vector are prevented from
circularizing. Linear molecules are transfected directly into COS-7 cells and then proc-
essed as described in the second basic protocol (beginning at step 10). Direct liga-
tion/transfection eliminates the need to subclone target fragments into pTAG4 and select
colonies in E. coli. The technique is capable of assaying, in equal representation, both
DNA strands of each individual restriction fragment of target DNA, regardless of size. It
is not compatible with the strategy and vector for trapping internal exons.
Additional Materials (also see Basic Protocol 2)
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock
solutions, see APPENDIX 2; for suppliers, see SUPPLIERS APPENDIX.
NruI restriction endonuclease
16°C water bath
1. Digest ≥20 µg pTAG4 with NruI and one of the restriction endonucleases that cut in
the pTAG4 multiple cloning site (see Fig. 6.1.3).
This digestion leaves a blunt end 3 kb upstream of the transcriptional start in the SV40
promoter and a single cloning site 50 to 300 bases downstream of the second exon of
pTAG4.
2. Purify the larger pTAG4 fragment (3.7 to 3.95 kb) by agarose gel electrophoresis and
the glass-bead method. Resuspend in TE buffer to final concentration of 0.5 µg/µl.
Other DNA purification methods can be substituted for the glass-bead method.
3. Digest ≥5 µg target DNA with the same restriction endonuclease chosen for digestion
of pTAG4 in step 1.
See second basic protocol, steps 5a to 8a and 5b to 8b. For YAC target DNA, digest ≥2 ìg.
Select a restriction endonuclease that cuts within the multiple cloning site (see Fig. 6.1.3).
Do not use NruI.
4. Phenol extract and ethanol precipitate digested target DNA. Resuspend in TE buffer
to a final concentration of 0.5 µg/µl.
5. Set up a ligation reaction as in step 5 of the first basic protocol except use digested,
purified pTAG4 DNA (step 2) in place of pSPL3 DNA and digested, purified target
DNA (step 4) in place of cosmid DNA. Incubate overnight at 16°C.
6. Transfect ligation reaction into COS-7 cells by lipid-mediated transfection (first basic
protocol, steps 10 to 18).
7. Prepare total RNA, digest it with DNase I, and perform RT-PCR to detect trapped 3′
terminal exons (second basic protocol, steps 10 to 25).

Identifying
Candidate Genes
in Genomic DNA

6.1.15
Current Protocols in Human Genetics Supplement 3
 pTAG4 target DNA
digest with
NruI/EcoRI digest restriction enzyme EcoRI digest
(alternate protocol,
steps 1-4)

NruI 5′ EcoRI EcoRI 3′ EcoRI

*
1 2
SV40
ligate
(basic protocol 1, step 5)

NruI 5′ 3′ 5′ NruI
*
1 2 2 1
SV40 SV40
transfect into COS-7 cells
(basic protocol 1, steps 10-18)

prepare RNA and carry out RT-PCR

(basic protocol 2, steps 10-25)
3′ terminal exon

Figure 6.1.5 Schematic representation of direct ligation/transfection. pTAG4 is double-digested

with NruI and one restriction enzyme cutting within the multiple cloning site. This effectively produces
an unligatable blunt end upstream of the promoter and leaves a sticky clonable end downstream
of viral exon 2 of pTAG4. Target DNA is digested with the same restriction enzyme used to generate
the sticky end downstream of viral exon 2 and ligated to digested pTAG4. This forms concatemers
of target restriction fragments flanked on each end by a single pTAG4. These ligation reactions are
directly transfected into COS-7 cells to induce transcription from the concatemers (arrow inidicates
the direction of transcription). Total RNA isolation and performed as in the second basic protocol.
Both strands of each restriction fragment from the target DNA are assayed equally, regardless of
fragment complexity or size. Abbreviations: SV40, SV40 early promotor; 1,2, adenovirus-2 leader
exons 1 and 2; *, poly(A) signal sequence.

SUPPORT CLONING PCR PRODUCTS WITH URACIL DNA GLYCOSYLASE

PROTOCOL Cloning PCR products is often desirable to establish a permanent source of cloned DNA
for hybridization studies, to obtain high-quality DNA sequencing results, or to separate
products when PCR amplification yields a complex mixture. This support protocol
describes a method for cloning secondary PCR products resulting from the exon-trapping
procedure (Fig. 6.1.6). This technique may also be utilized to rapidly and efficiently clone
any PCR-generated DNA, provided that appropriate primers are used. It requires synthesis
of oligonucleotide primers containing twelve specific nucleotides added at the 5′ end (i.e.,
5′-CUACUACUACUA-template specific sequence-3′). Four of the twelve additional
nucleotides are dU and provide targets for the enzyme uracil DNA glycosylase (UDG).
UDG treatment of PCR products made with dU-modified primers results in the creation
of 12-base 3′ single-stranded termini. UDG-treated DNA can be annealed to vector DNA
that has been generated using dU primers complementary to those used for target DNA.
Alternatively, the pAMP10 vector can be used; this is a commercially available vector
designed to accommodate cloning of UDG-treated DNA. Two advantages of the UDG-
Isolation of Exons cloning method are: (1) ease of cloning—no restriction enzyme digestions or ligations
from Cloned
DNA by Exon are required and the reaction is completed in 30 min, and (2) efficiency of cloning—1 ×
Trapping 107 clones per µg DNA are routinely achieved.
6.1.16
Supplement 3 Current Protocols in Human Genetics
 5 ′- 5′ 3′
CU dUSA4
AC 3′ AU
UA CA
CU + UC
AC AU
UA CA
3′ UC
-5 ′
dUSD2 3′ 5′

5 ′- CUA CUACUACUA TAGTAGTAGTAG-3 ′

3 ′- GATGATGATGAT A U C AUCAU C A UC- 5 ′

5 ′-
C_
AC
_A
C_
AC
_A
TAGTAGTAGTAG- 3 ′
3 ′- GATGATGATGAT A_
CA
_C
A_
CA
_C
-5 ′

exon DNA

3′ 3′
3′ 3′
SP6 T7

f1 IG plasmid
pAMP10 lacI

r
Ap ori
TA CA
TG TA

AT
AT

GT TC
GA C
AT TA

AG AT
TG TAC

TA CA
GT T C
GA AC

AG
CT

exon DNA

SP6 T7

f1 IG
lacI

Apr
ori

Figure 6.1.6 Schematic of uracil DNA glycolyase (UDG) cloning. Secondary amplification primers
dUSD2 and dUSA4 are annealed to DNA and amplified by PCR. pAMP10 (see Fig. 6.1.7) and uracil
DNA glycosylase (UDG) are added to vector DNA; UDG removes uracils and disrupts base-pairing,
exposing 3′ overhangs that then anneal to complementary pAMP10 vector ends. Annealing is done
for 30 min at 37°C. The resulting annealed molecules are used to transform competent cells.
Identifying
Candidate Genes
in Genomic DNA

6.1.17
Current Protocols in Human Genetics Supplement 3
 Additional Materials (also see Basic Protocol 1)
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock
solutions, see APPENDIX 2; for suppliers, see SUPPLIERS APPENDIX.
25 ng/µl pAMP10 plasmid supplied as a linear molecule (GIBCO/BRL; Fig. 6.1.7)
1 U/µl uracil DNA glycosylase (UDG)
Additional reagents and equipment for agarose gel electrophoresis (UNIT 2.7)
1. Add the following to a microcentrifuge tube:
1 to 2 µl (100 ng) PCR product
2 µl 25 µg/ml pAMP10 cloning vector
1 µl 10× Taq DNA polymerase buffer
1 µl 1 U/µl UDG
H2O to 10 µl final.
Mix and incubate 30 min at 37°C.
2. Transform 100 µl competent E. coli cells with 5 µl of the annealing reaction (from
step 1) and plate on LB/ampicillin plates. Incubate 16 hr at 37°C.
3. Pick a colony into 50 µl of the following solution in a 0.5-ml microcentrifuge tube
(one reaction tube per colony):
5 µl 10× Taq DNA polymerase buffer
1.5 µl 50 mM MgCl2
1 µl 10 mM 4dNTP mix in sterile H2O
1 µl 20 µM oligonucleotide dUSA4
1 µl 20 µM oligonucleotide dUSD2
0.5 µl 5 U/µl Taq DNA polymerase
Sterile H2O to 50 µl final.
If the second basic or alternate protocol was used, substitute oligonucleotides Ad2 and
UAP for dUSA4 and dUSA2.
This is a rapid method to determine the size of the PCR product that has been cloned.
4. Preheat thermal cycler to 94°C. Overlay sample with one drop mineral oil, place tube
into thermal cycler, and incubate 5 min at 94°C.
5. Carry out PCR using the following amplification cycles:
30 cycles: 45 sec 94°C (denaturation)
30 sec 55°C (annealing)
1 min 72°C (extension)
1 cycle: 10 min 72°C (extension)
Final step: indefinitely 4°C (hold).
6. Analyze 4 µl of PCR products on a 2% (w/v) agarose gel.
A faint 177-bp band results from amplification of pSPL3 sequences. Bands >177 bp may
be exons and should be analyzed further.

Isolation of Exons
from Cloned
DNA by Exon
Trapping

6.1.18
Supplement 3 Current Protocols in Human Genetics
 A lac OPZ′
1
Nae I 3987 Aat II 191 multiple cloning site (MCS)
DraIII 3881
PstI 289

BglI 6
BsaI FspI 17 BsaA1 208
3884 PvuI 37 NarI 561
SspI 3676 Afl III 203 Esp3 1591
NspHI 197
Bsp HI 3578 SP6 promoter T7 promoter
HpaI 696
SspI 3545
XmnI 3336 f1 intergenic region EcoRV 752
Eco 57I 3425
BcgI 3259 BssHII 789
ScaI 3221 Apr ApaI 993
pAMP10
PvuI 3110 4118 bp 1000
(linearized) BsgI 1134 Bst EII 1018
3000 FspI 2963 BclI 1186
BsgI 1334
BglI 2857
lacI
BssHII 1657
BspHI 2570
NspHI 1850 ori
AftIII 1850 pfl Ml 1619
Eam1105I 2738 Eco 57I 2377
Drd I 1952
lacI promoter

Alw NI 2261
2000
B
150 SP6 transcription start
M13/pUC 23 - base forward sequencing primer SP6 promoter primer
5′ CCCAGT CACGACGTTG TAAAACG 3′ 5′ ATTTAGG TGACACTATA G 3′
5′ CCCAGT CACGACGTTG TAAAACGACG GCCAGTGAAT TGAATTTAGG TGACACTATA GAAGAGCT AT
3′ GGGTCA GTGCTGCAAC ATTTT GCTGC CGGTCACTTA ACTTAAATCC ACTGTGATAT CTTCTC GATA
SP6 promoter
200 250
AatII SphI MluI SnaBI HindIII BamHI XbaI NotI cloning region
GACGTCGCAT GCACGCGTAC GTAAGCTTGG A T CCTCTAGA GCGGCCGCCT ACTACTACTA 3′
CTGCAGCGTA CGTGCGCATG CATTCGAACC T AGGAGATCT CGCCGGCG

SunI XmaIll

300
Kpn2I AgeI Sse 8387I
T7 promoter
TCGACCC G GGAATTCCGG ACCGGTACC T GCAGGCGTAC CAGCTTT CCC TATAGTGAGT C GTATTA GAG
3′ ATCATCATCA TCAGCTGGGC CCTTAAGGCC TGGCCATGGA CGTCCGCATG GTCGAAA GGG ATATCACTCA GCATAAT CTC
3′ GGG ATATCACTCA GCATAAT 5′
cloning region SalI SmaI EcoRI RsrI KpnI Pstl T7 promoter primer
AccI
T7 transcription start
350
CTTGGCGTAA TCATGGTCAT AGCTGTTTCC TGTGTGAAAT TGTATCCGCT 3′
GAACCGCATT AGTACCAGTA TCGACAAAGG ACACACTTTA ACAATAGCGA 5′
3′ GG ACACACTTTA ACAATAGCGA 5′
α - peptide start M13/pUC reverse sequen cing primer

Figure 6.1.7 Map of pAMP10 vector. (A) The multifunctional vector is derived from pSPORT 1 and contains T7 and
SP6 promoters, lac promoter and repressor sequences, a multiple cloning site (MCS; shown below the vector map),
ampicillin resistance (Apr), and the phage F1 intergenic region for preparation of single-stranded DNA. (B) The
sequence of the MCS cloning region is indicated; it contains 12-base 3′ termini that are complementary to PCR
products generated using oligonucleotide primers with 5′-CUACUACUACUA extensions—e.g., dUSD2 and dUSA4
secondary amplification primers.

6.1.19
Current Protocols in Human Genetics Supplement 3
 REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2; for suppliers, see SUPPLIERS APPENDIX.
DNase I buffer, 10×
200 mM Tris⋅Cl, pH 8.3
500 mM KCl
25 mM MgCl2
1 mg/ml BSA
Store several months at −20°C
First-strand buffer, 5×
250 mM Tris⋅Cl, pH 8.3
375 mM KCl
15 mM MgCl2
Store ≤1 year at −20°C
COMMENTARY
Background Information
Internal exon trapping
The exon-trapping procedure described in
the first basic protocol is a modification of a
method developed by Buckler et al. (1991).
Following publication of the original procedure
using the plasmid pSPL1, an updated version
of this vector, pSPL3, was constructed (Church
et al., 1994). With the pSPL3 vector, exons are
trapped directly from cloned genomic DNA by
splicing, using the ability of cells to recognize
and remove intron sequences from premes-
senger RNA molecules (Green, 1991). The
pSPL3 plasmid, an E. coli/COS-7 cell shuttle
vector, contains the HIV tat intron and flanking
exon sequences. A multiple cloning site
(MCS) was inserted into the HIV tat intron
allowing the cloning of foreign DNA into the
intron. The HIV tat sequences are flanked 5′
by the SV40 early promoter and origin of
replication (ori) , a n d 3′ b y t h e S V 4 0
polyadenylation signal (pA; see Fig. 6.1.2).
These vector components permit high levels
of replication and transcription of subcloned
DNA sequences transfected into COS-7 cells
(Gluzman, 1981). Exons will be trapped from
genomic DNA subcloned in pSPL3, provided
that the subclone contains an exon in the
proper orientation with both splice-acceptor
and splice-donor sites (see Fig. 6.1.1).
Trapped exons are identified following re-
verse transcription and polymerase chain reac-
tion (RT-PCR) amplification of cytoplasmic
RNA of transiently transfected cells using vec-
Isolation of Exons
from Cloned tor-specific oligonucleotides (see Fig. 6.1.1).
DNA by Exon During a second round of PCR, a nested set of
Trapping vector-specific primers provides additional speci-
6.1.20
Supplement 3
ficity. These secondary PCR primers contain dU
residues for cloning the PCR products using uracil
DNA glycosylase (UDG; Nisson et al., 1991).
The UDG cloning method outlined in Figure
6.1.6 was originally developed using a pair of
primers designed to amplify pUC119 vector
DNA
(5′ UAGUAGUAGUAGACCATCGTCGA-
CCTGCAG-3′ and 5′ UAGUAGUAGUA-
GACCATCGGATCCCCGGGT-3′). Treatment
of pUC119 vector DNA with UDG after PCR
amplification results in a vector that can rapidly
anneal to UDG-treated PCR DNA generated
with primers designed to install complemen-
tary ends (i.e., 5′- CUACUACUACUA exten-
sions). pAMP10 (Fig. 6.1.7) is a multifunc-
tional vector developed recently by these
authors for UDG cloning that eliminates the
requirement for preparation of vector DNA.
Exon trapping is a useful technique in a
positional cloning strategy to isolate candidate
disease genes. It has been used to isolate, among
others, the gene responsible for Huntington
disease (The Huntington Disease Research
Collaborative, 1993). As a gene-identification
method, it compares favorably to other ap-
proaches (e.g., zoo blots, HTF/CpG island
identification, and cDNA library screening) be-
cause it results in the direct isolation of gene
sequences from cloned genomic DNA without
prior knowledge of a gene’s structure or pattern
of expression. Similar approaches for trapping
exons have been developed by other investiga-
tors and, with the exception of the retroviral-
based system of Duyk et al. (1990), most share
common features (Auch and Reth, 1990; Ham-
aguchi et al., 1992).
The retroviral vector–based exon trapping
Current Protocols in Human Genetics
method of Duyk et al. (1990) permits the re-
covery of internal exons, although this ap-
proach suffers from several limitations. Three
cell lines are required and the procedure is
complicated and time consuming; the authors
state that twenty cosmids can be screened in 4
weeks. In addition, because splice acceptors are
selected by this method, partial exons may be
preferentially isolated, yielding a smaller hy-
bridization probe for downstream analysis.
Auch and Reth (1990) developed a simplified
system in which a COS–E. coli shuttle vector
was constructed using the RSV LTR promoter
and part of the rat preproinsulin gene as a source
o f splice-donor, splice-acceptor, and
polyadenylation sites. The authors demon-
strated that an exon can be trapped from a single
subclone but did not address the question of
how much DNA can be screened per transfec-
tion without a concomitant loss of sensitivity.
Hamaguchi et al. (1992) have also constructed
a COS–E. coli shuttle vector, pMHC2, that
apparently addresses this issue; they maintain
that six exons can be trapped from a pool of
subclones derived from eight cosmids or ∼300
kb of DNA. Because they do not know how
many exons the starting material contained, the
question of sensitivity remains unanswered.
3′ terminal exon trapping
The strategy outlined in the second basic
protocol for identification of 3′ terminal exons
from cloned genomic DNA sources is a modi-
fication of an earlier approach (Krizman and
Berget, 1993). The vector strategy is based on
earlier work suggesting that the exon is the unit
of recognition of the RNA-processing machin-
ery (Robberson et al., 1990). More recent re-
sults also suggest a very strict biochemical
definition of 3′ terminal exons which makes
them a likely candidate for this trapping ap-
proach (Niwa and Berget, 1991, 1992).
3′ terminal exon trapping uses the trapping
vector pTAG4 (see Fig. 6.1.3). This plasmid is
a pUC-based vector that harbors an incomplete
transcription unit consisting of the SV40 pro-
moter/enhancer region followed by leader ex-
ons 1 and 2 from the human adenovirus-2.
These nonprotein-coding exons are internal ex-
ons separated by the natural short intron. This
DNA virus transcription unit is incomplete be-
cause it lacks a 3′ terminal exon to direct
polyadenylation for production of mature, sta-
ble mRNA. The basis of this exon-trapping
approach is to ask the foreign DNA inserted
downstream of the second adenovirus exon to
donate a 3′ terminal exon and produce stable,
Current Protocols in Human Genetics
mature, chimeric mRNA that can be amplified
by the reverse transcriptase polymerase chain
reaction (RT-PCR) technique of 3′ rapid ampli-
fication of cDNA ends (3′ RACE; Frohman et
al., 1988; see Fig. 6.1.4).
The differences between trapping internal
exons and 3′ terminal exons should be consid-
ered. 3′ terminal exon trapping relies on posi-
tive selection. No RT-PCR product is produced
without the addition of a valid last exon to
pTAG4. Experiments designed to induce false-
positive scoring indicate a high degree of speci-
ficity of this system in scoring only valid last
exons (Krizman and Berget, 1993). There ap-
pears to be a very strict biochemical definition
of last exons. The great majority of vertebrate
genes contain a single last exon; thus the same
gene is not scored repeatedly, which would
complicate the analysis. Although the average
size of 3′ terminal exons is 627 bp, the majority
are in the 200- to 400-bp range, making the
RT-PCR product from this approach much
larger than the products resulting from the in-
ternal exon trapping approaches. This gives
greater sequence information per trapped exon.
The biochemical differences between inter-
nal and last exons provides some insight into
the possible reasons for greater selectability of
last exons versus internal exons. Internal exons
have an average size of 120 bp and usually
consist of an open reading frame bordered by
3′ and 5′ splice sites. The 5′ splice sites are
reasonably well conserved, but the consensus
sequence is relatively short and only the two
nucleotides at the splice junction are invariant.
The 3′ splice sites are poorly conserved with
the exception of the invariant dinucleotide at
the splice site. On the other hand, 3′ terminal
exons have an average size of 627 bp and
usually consist of a partial open reading frame
bordered at the 5′ end by a 3′ splice site. They
contain a highly conserved poly(A) signal that
is found 15 to 30 nucleotides upstream of the
cleavage/poly(A)-addition site. The poly(A)
signal consists of either the sequence
AAUAAA or AUUAAA. The presence of this
sequence is one of the defining hallmarks of a
last exon (Moore et al., 1992). Thus, the need
for a valid 3′ splice site in close proximity to
an invariant poly(A) signal most probably con-
tributes to the high degree of specificity of the
3′ terminal exon trapping approach.
Critical Parameters
Identifying
Internal exon trapping Candidate Genes
The most notable modifications to the origi- in Genomic DNA
6.1.21
Supplement 3
 nal method (Buckler et al., 1991) have been
made to the plasmid, formerly pSPL1 and now
called pSPL3 (Church et al., 1994). Major im-
provements have also been made in the meth-
ods for COS-7 cell transfection, RNA isolation,
and PCR product cloning (Nisson et al., 1993).
Plasmid improvements. Of the three im-
provements to the original pSPL1 plasmid, per-
haps the most important reduces undesirable
products and increases the complexity of DNA
that can be transfected per tissue culture dish.
With the original method, pSPL1-derived RNA
without a trapped exon comprised a large frac-
tion of the molecules generated after sub-
cloning and transfection. This undesirable
product consumed most of the PCR compo-
nents, thus limiting the sensitivity of the sys-
tem. Using pSPL3, subclones that contain par-
tial or alternatively spliced exons, exons in the
reverse orientation, introns, or no insert will
result in vector-only molecules. When vector-
only mRNAs are generated, converted into
cDNAs, and made double-stranded, the two
BstXI half-sites adjacent to the vector splice-
donor and splice-acceptor sites form a BstXI
site that can be cut to greatly reduce the entry
of vector-only molecules into secondary PCR.
This permits higher-complexity DNA to be
used per transfection than with the original
pSPL1 vector (Buckler et al., 1991), and allows
molecules containing trapped exons to be am-
plified and visualized after gel electrophoresis
(see Fig. 6.1.1). In other words, ten cosmids-
worth of DNA (∼400 kb) can be screened per
transfection using pSPL3 compared with
pSPL1 (∼40 kb).
A second problem with pSPL1 was the use
of a cryptic splice-donor site located to the right
of the cloning site. Normally, when an exon is
trapped, the MCS is spliced out, unless a cryptic
splice-donor site located 3′ to the MCS is used
(see Fig. 6.1.1). Formation of cryptic splice-do-
nor molecules, which occurred with pSPL1 and
can occur with pSPL3, is greatly reduced by
BstXI digestion. Use of the cryptic splice-donor
can be diagnosed by the presence of a unique
restriction site, NdeI, located 16 bp 5′ of the
donor. This NdeI site is preserved when the
cryptic donor is used (see Fig. 6.1.1). Although
the cryptic splice-donor site is also present in
pSPL3, the amplification of molecules resulting
from its use can be greatly reduced by BstXI
digestion. There are BstXI sites flanking the
MCS; these sites are preserved in the cryptic
Isolation of Exons splice-donor users (see Fig. 6.1.2). These can be
from Cloned
DNA by Exon inactivated by BstXI digestion, thus preventing
Trapping their amplification in secondary PCR.
6.1.22
Supplement 3
A third improvement to the plasmid is the
replacement of the single BamHI cloning site
with an MCS. There are unique restriction en-
donuclease sites within the MCS of pSPL3 for
EcoRI, SstI, XhoI, NotI, XmaIII, PstI, BamHI,
and EcoRV, allowing a great deal of flexibility
in experimental design. Although a combina-
tion of BamHI and BglII single- and double-di-
gests of cosmid DNA cloned into the BamHI
site of pSPL3 generally yields good results,
other sites work equally well. Subcloning into
EcoRI or EcoRV sites will inactivate the over-
lapping BstXI sites and eliminate their useful-
ness in reducing the recovery of cryptic splice-
donor users as previously discussed.
Shotgun subcloning genomic DNA into
pSPL3. The efficiency of trapping exons from
cloned genomic DNA—e.g., cosmid, phage, or
yeast artificial chromosome (YAC) clones—
depends on the overall representation of target
DNA sequences subcloned into pSPL3. Be-
cause a pSPL3 subclone must contain an entire
internal exon in order to capture it, restriction
sites within an exon may prevent its capture if
they are recognized by the subcloning restric-
tion enzyme. Several strategies can be used to
ensure a high level of representation of genomic
DNA, including combining digestions from
more than one restriction enzyme or gel isola-
tion and subcloning specific restriction frag-
ments. Also, it is important to monitor vector
background (religated pSPL3) during sub-
cloning to make sure that >90% of resultant
colonies are true recombinants.
Trapping exons from YACs presents a spe-
cial problem because YACs are difficult to com-
pletely separate from contaminating yeast
DNA. This problem can be minimized by care-
fully cutting the YAC band from a pulsed-field
gel and running that band on a second gel;
however, if the YAC DNA migrates close to a
yeast chromosome, contamination with yeast
sequences may be unavoidable. Several of the
trapped exons in this case may be yeast se-
quences. Yeast sequences can be identified by
hybridization to a filter containing yeast chro-
mosomes or digested total yeast DNA.
Transfection of COS-7 cells. Although elec-
troporation was used in Buckler’s exon trap-
ping protocol (Buckler et al., 1991), cationic
lipid transfection (CPMB UNIT 9.4) has been found
to be more convenient for transfecting animal
cells (Duyk et al., 1990; Hawley-Nelson et al.,
1993). Transfection using cationic lipids is
much less expensive in time and reagents than
electroporation. Also, multiple transfections are
easily carried out using tissue culture dishes
Current Protocols in Human Genetics
with six 3.5-cm wells. It is important to opti-
mize the amount of cationic lipid used because
there is lot-to-lot variation. Other critical pa-
rameters include the amount of DNA that is
transfected, the percent confluence of the COS-
7 cells, and the method of DNA–cationic lipid
complex preparation. For a typical transforma-
tion, 3 to 6 µl cationic lipid (LipofectACE)
should be used with 0.5 to 1 µg DNA and
complexes should be formed for 10 min.
Preparation of total RNA. The rapid
guanidinium method to isolate total RNA (UNIT
10.4 & CPMB UNIT 4.2) increases the number of
samples that can be processed at a time and
consistently produces RNA of the highest qual-
ity and quantity (Chomczynski and Sacchi,
1987). However, any standard RNA-isolation
protocol can be used to obtain RNA for exon
trapping.
First-strand cDNA synthesis. It is important
not to add too much RNA to the first-strand
cDNA synthesis reaction because aberrant
products will result. The recommendation of 1
to 3 µg RNA for every 200 U reverse transcrip-
tase should be followed. SuperScript II and
AMV reverse transcriptases produce compara-
ble results.
Primary and secondary PCRs. See the Com-
mentary in CPMB UNIT 15.1 for a discussion of the
critical parameters for PCR amplification.
The protocols in this unit require that all
reaction components except Taq DNA po-
lymerase be heated 5 min at 94°C before
adding Taq DNA polymerase; this “hot start”
procedure improves the productivity of the
reaction.
The secondary PCR primers dUSA4 and
dUSD2 each contain twelve nucleotides (CAU-
CAUCAUCAU) at the 5′ end that allow the
PCR products to be cloned using uracil DNA
glycosylase (support protocol). Only portions
of secondary PCR primers hybridize specifi-
cally to the template (for dUSA4, 5′ CTGA-
GGAGTGAATTGGTCG-3′; for dUSD2, 5′
GTGAACTGCACTGTGACAAGCTGC-3′).
The primers can also be modified to allow PCR
products to be cloned by other methods—e.g.,
by adding nucleotides at 5′ ends to install spe-
cific restriction endonuclease sites for cloning.
3′ terminal exon trapping
Restriction endonuclease digestion and pu-
rification of both pTAG4 and target DNA from
the various types of clones discussed pre-
viously are critical. pTAG4 must be cut to
completion and gel-purified before being re-
suspended in TE buffer in step 4 of the second
Current Protocols in Human Genetics
basic protocol. When using linearized pTAG4
in the alternate protocol, the vector must be gel
purified to remove the multiple cloning site
fragment. Although it is not essential to purify
the linearized vector for the second basic pro-
tocol, it is generally a good idea to purify the
vector whenever subcloning is performed, to
minimize background. Target DNA must be
prepared in large enough quantity for further
manipulations. YAC DNA must be prepared
according to the procedure discussed. The
problems associated with genomic yeast DNA
are the same as those encountered in internal
exon trapping (see Critical Parameters for shot-
gun subcloning).
The method of presenting target DNA to
pTAG4—either shotgun cloning (second basic
protocol) or direct ligation/transfection (alter-
nate protocol)—must be decided. Shotgun
cloning has the advantage of producing a large
amount of recombinant DNA for multiple
transfections. Furthermore, subcloning meth-
ods are widely used in many laboratories. Fa-
miliarity with the technique can prove useful
should the need for troubleshooting arise. In
direct ligation/transfection, on the other hand,
there are no controls to indicate if the reaction
has failed, and failure cannot be detected until
several additional steps have been completed.
Direct ligation/transfection enjoys several ad-
vantages over shotgun cloning. First, it by-
passes the cloning bias of E. coli, which mani-
fests itself by the fact that often fragments of
DNA simply cannot be cloned into a vector
because of the size or sequence of the insert, or
for many other unforeseen reasons. Therefore,
it may be preferable to use direct ligation when
attempting to trap exons from targets with
higher complexity. Secondly, as seen in Figure
6.1.5, direct ligation/transfection is capable of
forming concatemers of DNA consisting of
each restriction fragment from the target DNA
flanked on each side by a single copy of pTAG4.
The result is that when these concatemers are
transfected into COS-7 cells, both strands of
each restriction target fragment are assayed
equally, regardless of size or complexity. Also,
direct ligation/transfection saves days in clon-
ing and purification of vector recombinants.
The net result is that this approach saves time
and money, increases the possibility of assaying
fragments that would not have been cloned into
pTAG4 if shotgun cloning were used, and
avoids the requirement that an insert be ligated
in the correct orientation within recombinant
Identifying
vector. Candidate Genes
DNase I treatment of the RNA preparation in Genomic DNA
6.1.23
Supplement 3
 is of the utmost importance. If there is any
vector-derived DNA present in the RNA sam-
ple, there is potential for reverse transcription
of A-rich regions of this DNA, which will
produce unwanted PCR products that will not
represent spliced, polyadenylated sequences.
RT-PCR parameters are also very important.
The technique works equally well whether re-
verse transcribing 5 µg total RNA or 1 µg
oligo(dT)-selected mRNA from transfected
COS-7 cells. However, the annealing tempera-
ture must be 55°C because of the calculated Tm
and G+C base content of the primers SV40P,
Ad2, and UAP. The final MgCl2 concentration
must be 1.25 mM for the same reasons. If either
of these parameters are off, there will be re-
duced or no PCR product.
Use of hot-start PCR is required. Addition
of Taq DNA polymerase to each PCR reaction
should take place during the 80°C incubation,
after the reaction has first been heated to 94°C
for 3 min. This prevents false priming to non-
specific sequence. The SV40P, AP, and UAP
primers were designed specifically for 3′
RACE, so that the poly(A) tail is used on the 3′
end of the transcripts and the viral sequences
are used on the 5′ end of the transcripts. Use of
viral-sequence primers gives the utmost speci-
ficity to the PCR reactions.
Each trapping experiment that generates a
PCR product should be further evaluated by
sequencing the products for confirmation of a
trapped exon. This can be accomplished either
by gel purification of individual ethidium bro-
mide–stained bands followed by subcloning
into a vector for sequencing, or by direct sub-
cloning of the product from the PCR reaction
tube followed by further selection of subclones
to identify which ones will be used for sequenc-
ing. In gel purification of a single band for
subcloning, it becomes necessary to sequence
multiple subclones because many 3′ terminal
exons are the same size, and a single band does
not always indicate a single 3′ terminal exon.
Likewise, it becomes necessary to sequence
multiple subclones when subcloning directly
from the PCR reaction tube because many sub-
clones will contain inserts of different sizes.
Insert size of the subclones can be determined
by PCR analysis of individual subclones using
the original primers for secondary PCR (Ad2
and UAP).
A splicing event can be detected by compar-
ing vector sequences in the resulting PCR prod-
uct (Fig. 6.1.8) with those in the unspliced
vector (see Fig. 6.1.3). If EcoRI is used to
present target DNA to pTAG4, sequence anal-
ysis of the resulting PCR product should indi-
cate that those specific nucleotides defining the
5′ splice site of vector exon 2 are present,
followed directly by the newly trapped se-
quence (see sequence above the diagrammed
PCR product in Fig. 6.1.8). Thus, intron se-
quences from pTAG4 will have been spliced

poly(A) signal

AATAAA
or
ATTAAA

– – – – – – CGGCCTCCGAACG / NNNNNNNNNNNNN – – – – – – – – –
5′ Ad2 2 A UAP 3′
n
– – – – – – ACGTCGACCTGAG / NNNNNNNNNNNNN – – – – – – – – –

Figure 6.1.8 Schematic representation of anticipated results of sequence analysis of a hypotheti-

cal trapped 3′ terminal exon. Vector-derived residual sequence (adenovirus-2 leader exon 2) is
generated from splicing vector exon 2 to trapped 3′ terminal exon followed by RT-PCR using primers
specific for these vector sequences. If EcoRI is used to digest vector and target DNA, the sequence
should correspond to that shown above the diagrammed PCR product. The slash between the G
and N nucleotides represents the splice junction, where nucleotides on the 5′ side of the junction
are vector-derived and nucleotides labeled N are from the newly trapped 3′ terminal exon. If another
restriction enzyme from the multiple cloning site is used, a different splice site will be used and the
resulting residual vector-derived sequence will correspond to that shown below the diagrammed
Isolation of Exons PCR product. The sequence of the trapped 3′ terminal exon is represented by the shaded area.
from Cloned
DNA by Exon The sequence of the PCR product should be flanked at the 5′ end by the sequence of the PCR
Trapping primer Ad2 and on the 3′ end by the sequence of the PCR primer UAP. Abbreviation: (A)n, poly(A)
sequence.
6.1.24
Supplement 3 Current Protocols in Human Genetics
out. If an enzyme other than EcoRI is used, an
alternative 5′ splice site can be used at low
frequency (∼10%). This splice site (arrowhead,
see Fig. 6.1.3) is derived from the multiple
cloning site of pTAG4 (see Fig. 6.1.3), and the
nucleotides that will be directly upstream from
the newly trapped sequence will be different
(see sequence below the diagrammed PCR
product in Fig. 6.1.8). Sequence analysis of a
PCR product resulting from 3′ exon trapping
must show evidence that a valid 3′ terminal
exon is present before further analysis is carried
out.
If a splicing event does not occur, much of
the multiple cloning site derived from the vec-
tor will not have been spliced out and those
sequences will be present downstream of the
vector-derived splice junction and any new se-
quence. PCR products that have not spliced, yet
contain new sequence, most probably represent
reverse transcription of A-rich regions in pre-
cursor (nonspliced) RNA derived from vector-
driven transcripts. This problem can be over-
come either by making cytoplasmic RNA
preparations of transfected COS-7 cells or by
obtaining mRNA by oligo(dT) selection (CPMB
UNIT 4.5) to use for RT-PCR.
As far as has been determined, 3′ terminal
exon trapping works equally well with cosmid,
P1, bacteriophage, plasmid, or YAC clones.
Efficiency of trapping from these various
clones has yet to be determined. It is difficult
to predict the relative number of genes present
in different types of clones; thus, more work
needs to be done to give an indication of how
many genes score positive and how many are
missed. However, the larger and more complex
clones such as P1 and YAC clones may contain
many genes. There may be many ethidium
bromide–stained bands on the agarose gel
when RT-PCR results from trapping of these
clones are analyzed. It may be necessary to
subclone directly from the PCR reaction tube
and then sequence many subclones of different
insert sizes to get an idea of the representation
of trapped exons from these experiments.
Anticipated Results
Internal exon trapping
The first basic protocol results in cloned
internal exons (exons flanked by splice-ac-
ceptor and splice-donor sites). Initial, terminal,
or alternatively spliced exons cannot be cap-
tured with this method (Andreadis et al., 1993).
Although this protocol is designed for use with
cosmid clones of genomic DNA, exons have
Current Protocols in Human Genetics
been trapped from YAC, plasmid, and bacterio-
phage λ clones. The expected yield is one exon
trapped per one to two cosmids assayed using
the basic protocol, although this may vary de-
pending on the gene-richness of the region of
interest.
If individual cosmids are being subcloned,
one can reasonably handle 10 to 20 cosmids in
one experiment; however, if pooling strategies
are employed, more than 100 cosmids can be
processed simultaneously.
Undesirable products should be expected.
For example, ∼5% to 10% of the clones ob-
tained will contain sequence from the cloning
vector (e.g., cosmid vector), and about the same
proportion of clones will come from the HIV
tat intron. After sequencing, candidate exons
can be E-mailed via Internet to the BLAST
server (blast@Ncbi.NIH.gov) to scan the nucleic
acid data base (Altschul et al., 1990). This will
determine if the clones are HIV- or vector-de-
rived, as well as whether there is a match in the
database. Alternatively, exons >100 bp can be
E-mailed to the GRAIL server (grail@onrl.gov;
Uberbacher and Mural, 1991), which identifies
open reading frames (ORFs). If this is success-
ful, the ORF can be translated and E-mailed to
the protein database of the BLAST server.
Ultimately, proof of expression relies upon
trying one of several additional strategies:
northern blotting of the putative exon against
RNAs from a variety of tissues, RT-PCR of
RNA from a variety of sources, or—in a related
procedure—PCR amplification from primary
cDNA or cloned cDNA from a variety of
tissues.
3′ terminal exon trapping
3′ terminal exon trapping results in PCR
products that consist of partial vector-derived
sequence from the second exon, new sequence
from the trapped 3′ terminal exon, and the
sequences involved in 3′RACE (i.e., 17 adenine
residues and the primer sequence from UAP).
The largest sequence should be that of the
trapped exon. There should be a poly(A) signal
15 to 30 nucleotides upstream of the poly(A)
tail and evidence of a splice event upstream of
the new trapped sequence (Fig. 6.1.8). If a
splicing event has occurred and there is a
poly(A) signal present in the correct region of
the new sequence, then the sequence should be
considered a trapped 3′ terminal exon and fur-
ther analysis of the gene performed.
The results expected from both 3′ terminal Identifying
exon trapping protocols will be similar when Candidate Genes
assaying target DNA of limited complexity in Genomic DNA
6.1.25
Supplement 3
 (e.g., plasmid clones or single-fragment in-
serts). However, when the target DNA is of
greater complexity (e.g., cosmid clones, P1
clones, pooled cosmid clones, and YACs), the
resulting trapping efficiency will be greater if
direct ligation/transfection (alternate protocol)
is used. With the alternate protocol, there is no
subcloning of restriction fragments from com-
plex DNA sources into pTAG4 and subsequent
tranformation of E. coli. This eliminates the
possibility that certain unclonable restriction
fragments will not be assayed. Thus, with the
alternate protocol all restriction fragments in a
complex DNA source will be assayed equally,
which may not be the case with the shotgun
cloning protocol.
3′ terminal exons should contain short open
reading frames at the 5′ end of the exon. The
majority of the sequence, most likely, will be
noncoding, and many stop codons may be
found very near the 5′ end in all three reading
frames. The ultimate proof of the existence of
a trapped 3′ terminal exon lies in detecting its
expression by northern blot or RT-PCR analy-
sis, as well as its expression as a cDNA clone.
These trapped exons make extremely specific
and strong probes for screening cDNA librar-
ies.
Time Considerations
For internal exon trapping, preparation of
the vector and shotgun subcloning of cosmid
DNAs take 2 days. One day is needed to
miniprep DNAs and transfect COS cells. Tran-
sient expression requires one day, at the end of
which RNA can be prepared. First-strand
cDNA synthesis, primary PCR, and BstXI di-
gestion take 1 day. Secondary PCR, running the
analytical gel, and starting the UDG cloning
protocol take 1 day. Colony PCR of a selected
group of clones requires 1 day. The minimum
total amount of time required for the basic and
support protocols is 7 to 8 days.
3′ terminal exon trapping with shotgun sub-
cloning takes roughly the same amount of time
as internal exon trapping to obtain subclones
ready for sequencing, ∼7 days. See UNIT 5.7 for
the time considerations when isolating YAC
DNA from a pulsed-field gel. Direct liga-
tion/transfection avoids subcloning and mini-
prep steps, thus saving two days.
Isolation of Exons
from Cloned
DNA by Exon
Trapping
6.1.26
Supplement 3
Literature Cited
Altschul, S.F., Gish, W., Miller, W., Myers, E.W.,
and Lipman, D.J. 1990. Basic local alignment
search tool. J. Mol. Biol. 215:403-410.
Andreadis, A., Nisson, P.E., Kosik, K.S., and Wat-
kins, P.C. 1993. The exon trapping assay partly
discriminates against alternatively spliced exons.
Nucl. Acids. Res. 21:2217-2221.
Auch, D. and Reth, M. 1990. Exon trap cloning:
Using PCR to rapidly detect and clone exons
from genomic DNA fragments. Nucl. Acids Res.
18:6743-6744.
Buckler, A.J., Chang, D.D., Graw, S.L., Brook J.D.,
Haber, D.A., Sharp, P.A., and Housman, D.E.
1991. Exon amplification: A strategy to isolate
mammalian genes based on RNA splicing. Proc.
Natl. Acad. Sci. U.S.A. 88:4005-4009.
Chomczynski, P. and Sacchi, N. 1987. Single-step
method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform extraction.
Anal. Biochem. 162:156-159.
Church, D.M., Stotler, C.J., Rutter, J.L., Murrell,
J.R., Trofatter, J.A., and Buckler, A.J. 1994. Iso-
lation of genes from complex sources of mam-
malian DNA using exon amplification. Nature
Genet. 6:98-105.
Duyk, G.M., Kim, S., Myers, R.M., and Cox, D.R.
1990. Exon trapping: A genetic screen to identify
candidate transcribed sequences in cloned mam-
malian genomic DNA. Proc. Natl. Acad. Sci.
U.S.A. 87:8995-8999.
Frohman, M.A., Dush, M.A., and Martin, G.R.
1988. Rapid production of full-length cDNAs
from rare transcripts: Amplification using a sin-
gle gene-specific oligonucleotide primer. Proc.
Natl. Acad. Sci. U.S.A. 85:8998-9002.
Gluzman, Y. 1981. SV40 transformed simian cells
support the replication of early SV40 mutants.
Cell 23:175-182.
Green, M. 1991. Biochemical mechanisms of con-
stitutive and regulated pre-mRNA splicing. Ann.
Rev. Cell Biol. 7:559-599.
Hamaguchi, M., Sakamoto, H., Tsuruta, H., Sasaki,
H., Muto, T., Sugimura, T., and Terada, M. 1992.
Establishment of a highly sensitive and specific
exon-trapping system. Proc. Natl. Acad. Sci.
U.S.A. 89:9779-9783.
Hawley-Nelson, P., Ciccarone, V., Gebeyehu, G.,
Jessee, J., and Felgner, P.L. 1993. LIPOFEC-
TAMINE reagent: A new, higher efficiency polyca-
tionic liposome transfection reagent. Focus 15:73-
79.
The Huntington’s Disease Research Collaborative.
1993. A novel gene containing a trinucleotide
repeat that is expanded and unstable on Hunt-
ington’s Disease chromosomes. Cell 72:971-
983.
Krizman, D.B. and Berget, S.M. 1993. Efficient
selection of 3′-terminal exons from vertebrate
DNA. Nucl. Acids Res. 21:5198-5202.
Current Protocols in Human Genetics
Nisson, P.E., Rashtchian, A., and Watkins, P.C.
1991. Rapid and efficient cloning of Alu-PCR
products using uracil DNA glycosylase. PCR
Methods Appl. 1:120-123.
Nisson, P.E., Hadley, R.G., and Watkins, P.C. 1993.
Finding and cloning genes directly from com-
plex DNA by exon trapping. Focus 15:26-31.
Niwa, M. and Berget, S.M. 1991. Mutation of the
AAUAAA polyadenylation signal depresses in
vitro splicing of proximal but not distal introns.
Genes & Dev. 5:2086-2095.
Niwa, M. and Berget, S.M. 1992. Are vertebrate
exons scanned during splice-site selection? Na-
ture 360:277-280.
Robberson, B.L., Cote, G.J., and Berget, S.M. 1990.
Exon definition may facilitate splice site selec-
tion in RNAs with multiple exons. Mol. Cell.
Biol. 10:84-94.
Uberbacher, E.C. and Mural, R.J. 1991. Locating
protein-coding regions in human DNA se-
quences by a multiple sensor-neural network
approach. Proc. Natl. Acad. Sci. U.S.A.
88:11261-11265.
Key Reference
Church et al., 1994. See above.
Describes use of the internal exon trapping method
for cosmids.
Green, M. 1991. Biochemical mechanisms of con-
stitutive and regulated pre-mRNA splicing. Ann.
Rev. Cell Biol. 7:559-599.
Very good review of the splicing literature to help
understand the basis behind the ability of pTAG4 to
trap a 3′ terminal exon and pSpL3 to trap an internal
exon.
Krizman and Berget, 1993. See above.
Original report of identifying genes from fragments
of genomic DNA by 3′ terminal exon trapping.
Contributed by Paul E. Nisson (internal
exon trapping)
Life Technologies
Gaithersburg, Maryland
Paul C. Watkins (internal exon trapping)
Sequana Therapeutics
La Jolla, California

David B. Krizman (3′ terminal exon trapping)

National Center for Human Genome
Research
Bethesda, Maryland

Identifying
Candidate Genes
in Genomic DNA

6.1.27
Current Protocols in Human Genetics Supplement 3