- Email: [email protected]
RACE: RAPID AMPLIFICATION OF cDNA ENDS
4 RACE: RAPID AMPLIFICATION OF cDNA ENDS Michael A. Frohman
Although the technology of c D N A cloning has been steadily grow ing more powerful (Gubler and Hoffman 1983; Heidecker and Mes sing 1987; Efstratiadis et al 1977} Okayama et al 1987; Coleclough 1988), the generation of full-length c D N A copies of m R N A tran scripts is often challenging, particularly with respect to obtain ing the 5' ends of messages. T h e first step, reverse transcription of mRNA, is usually the limiting one, and many variations have been described, including the use of different types of reverse transcrip tase, pretreatment of the m R N A to decrease secondary structure, al teration of reverse transcriptase conditions, and the use of primerextended c D N A instead of oligo(dT)-primed reverse transcription. Such modifications can profoundly affect the likelihood of obtaining full-length c D N A clones, but optimal conditions for reverse tran scription of a given m R N A are rarely established, since weeks to months are required to prepare and screen a single c D N A library and to isolate and analyze candidate cDNAs. The method described here, known as the Rapid Amplification of c D N A Ends (RACE) protocol (Frohman et al 1988), differs markedly in that the analysis of the reverse transcribed products can be per formed within 1 to 2 days of the start of the experiment. T h e m R N A 28
PCJR Protocols: A Guide to Methods and Applications Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
4 . RACE: Rapid Amplification of c D N A Ends
29
reverse transcription step can be modified and repeated until it is clear that the desired c D N A has been produced. Furthermore, infor mation about alternate splicing and promoter use is obtained, and the RACE protocol can be used to separate c D N A s produced from different transcripts prior to cloning steps. Finally, RACE products can be efficiently cloned into standard high-copy plasmid vectors, allowing many independent isolates to be obtained from a single plate of transformants. In essence, the RACE protocol generates cDNAs by using P C R to amplify copies of the region between a single point in the transcript and the 3 ' or 5' end. To use the RACE protocol, one must know a short stretch of sequence from an exon. From this region, primers oriented in the 3 ' and 5 ' directions are chosen that will produce overlapping c D N A s when fully extended. These primers provide specificity to the amplification step. Extension of the c D N A s from the ends of the messages to the specific primer sequences is accom plished by using primers that anneal to the natural (3' end) or a syn thetic (5' end) poly(A) tail. Finally, the overlapping 3 ' - and 5'-end RACE products are combined to produce an intact full-length c D N A . Figure 1 illustrates this strategy in more detail. In brief, for the 3 ' end, m R N A is reverse transcribed using a "hybrid" primer consist ing of oligo(dT) (17 residues) linked to a unique 17-base oligonucleo tide ("adapter") primer. Amplification is subsequently performed using the adapter primer, which binds to each c D N A at its 3 ' end, and a primer specific to the gene of interest. For the 5 ' end, reverse transcription is performed using a gene-specific primer. A homopolymer is then appended by using terminal transferase to tail the first-strand reaction products. Finally, amplification is accom plished using the hybrid primer previously described and a second gene-specific primer upstream of the first one. Additional factors considered in the development of the RACE protocol are described elsewhere (Frohman et al. 1988).
RACE Protocol
Materials
+
RNA: 1 f i g of poly(A) RNA is ideal; o n e can use as little as 200 ng of total RNA without modifying the procedure.
Part O n e . Basic M e t h o d o l o g y
30
RACE:
3' end
5' end
RACE:
mRNA VWVWWWVWvVM/WVWW <{2
cDNA
(-)
cDNA
7
I denature anneal
STRAND
« + j ( - ) STRAND
|5RTl
I remove excess 5RT primer
primer[3 « m p |
Uil cDNAs with dATP T
|3'»mp|
( • ) STRAND
t~> 8TRAN»
AAAAAAAARAI
r »
ITTTT**»*I
7
denature anneal primer fr***! xtend
STRAND gttlf i denature anneal primer |****TTTTl extend T
( +)
,TTTT| AAAAAAAAAAI
|?'»mpl
; (TO ^ R A N D "xls
(4> STRANP
TR
40 cycles
STRAND
denature anneal primer |5'»mp|
( - ) STRAND \*T*
I denature I anneal primers: I3'»n»pl and extend
( +) ^
STRAND
T R ( - ) STRAND | 5 ' . m p |
up to 6 • 10
|3+«n»pl copies
TR
7
denature anneal primer |»***j extend
(+) STRAND" £ - ) STRAND
of cDNA T R ( + ) STRAND I
}-¥•
T R C - } STRAND
Primers: ****
T -adapter: T _ dX 1X7 X
5'
I denature I anneal primers: \****\ and | S ' i m p |
GACTCGAGTC-
GACATCGATTTTTTTTTTTTTTTTT 3' T h i s sequence Clal
c o n t a i n s t h e Xho\,
Sail,
and
extend
recognition sites.
•
a
* * * * _ d a p t e r : 5' GACTCGAGTCGACATCG 3 ' 3'
4 0 cycles
up to
A M P — s p e c i f i c t o gene of interest, c o m p l e F***4
m e n t a r y t o ( - ) strand. 5RT
and 5 ' A M P — s p e c i f i c t o gene of interest,
I
T R ( » ) STRANP
I
6
T R < ~ ) STRAND |5TS71
- 10
Figure 1
S c h e m a t i c r e p r e s e n t a t i o n o ft h e RACE p r o t o c o l . O p e n r e c t a n g l e s
D N A strands actively b e i n g synthesized; shaded rectangles represent D N A synthesized. A t each
step t h ediagram
copies
of cDNA
c o m p l e m e n t a r y t o ( + ) strand.
represent previously
is s i m p l i f i e d t o i l l u s t r a t e o n l y h o w
the n e w
p r o d u c t f o r m e d d u r i n g t h e p r e v i o u s s t e p is u t i l i z e d . A ( - ) o r ( + ) s t r a n d is d e s i g n a t e d as " t r u n c a t e d " (TR) w h e n i t is s h o r t e r t h a n t h e o r i g i n a l ( - ) o r ( + ) s t r a n d , r e s p e c t i v e l y .
1 0 x RTC buffer: 500 mMTris-HCI (pH8.15) at41°C, 60 mMMgCI 2, 400 mM KCI, 10 mM DTT, each dNTP at 10 mM Hybrid dT 1-adapter primer: 7 GACTCGAGTCGACATCG AI I I I I I I I I I I I I I I I I—stock (1 /xg/ml) Adapter primer: GACTCGAGTCGACATCG Gene-specific primers: see Fig. 1
4 . RACE: Rapid Amplification of cDNA Ends
31
1 0 x PCR buffer: 670 mM Tris-HCI (pH 8.8 at room temperature), 67 mM MgCI 2, 1.7 mg/ml of bovine serum albumin, 166 mM ( N H 4) 2S 0 4 TE: 10 mM Tris-HCI (pH 7.6), 1 mM ETDA.
Amplification of cDNA 3 ' Ends Reverse Transcription 1. Assemble on ice: 2 /xl 1 0 x RTC buffer, 10 units of RNasin (Promega Biotech), 0.5 /xg of dT 1-adapter primer, and 10 units of 7 AMV reverse transcriptase (Life Sciences) in a total volume of 3.5 / x l . 2. Heat RNA in 16.5 /xl of H 20 for 3 minutes at 65°C; quench on ice and add to above. 3. Incubate at 42°C for 1 hour, and then at 52°C for 30 minutes. Dilute to 1 ml with TE and store at 4°C ("cDNA pool"). Amplification 1. Prepare PCR cocktail
5 /xl 1 0 x PCR Buffer 5 /xl DMSO 5 /xl 1 0 x dNTPs (15 mM each) 30 /xl H 20 1 /xl adapter primer (25 pmol//xl) 1 /xl gene-specific primer (25 pmol//xl) 1 to 5 /xl cDNA pool
2. Denature at 95°C for 5 minutes; cool to 72°C. Add 2.5 units of Taq polymerase and overlay with 30 /xl of min eral oil (heated to 72°C). Anneal at 55°C for 5 minutes; extend at 72°C for 40 minutes. Cycle: 95°C for 40 seconds / 55°C for 1 minute / 72°C for 3 minutes—for 40 cycles. Extend 72°C for 15 minutes.
Amplification of cDNA 5 ' Ends 1. Reverse transcribe RNA as previously described, but substitute 10 pmol gene-specific primer 1 for dT 1-adapter primer. 7
32
Part One. Basic M e t h o d o l o g y
2. Remove excess primer by using a Centricon 100 spin filter (Amicon Corp.). Dilute reverse transcription mixture with 2 ml of 0.1 x TE and centrifuge at 1000 x g for 20 minutes. Repeat and collect retained liquid. Concentrate to 10 /xl using Speed Vac centrifugation. 3. Add 4 /xl of 5 x Tailing Buffer (Bethesda Research Laboratories, Inc.), 4 /xl of 1 mM dATP, and 10 units of Terminal d Transferase (BRL). Incubate 5 minutes at 37°C, and then 5 minutes at 65°C. Dilute to 500 /xl with TE. 4. Amplify 1 to 10 /xl of cDNAs as previously described, adding dT 1-adapter primer (10 pmol) in addition to the adapter primer 7 and gene-specific primer 2.
Analysis Perform a Southern blot analysis of the amplification products. Test for single-stranded DNA by removing the PCR buffer (Centricon fil ter [any type], spun column, or ethanol precipitation) and incubat ing in mung bean nuclease buffer (BRL) and 1 unit of mung bean nu clease for 30 minutes at 30°C (Frohman et al. 1988). Compare to the samples incubated in buffer alone and in TE.
Cloning RACE Products Blunt Cloning Remove the PCR buffer and excess primer by three rounds of Cen tricon 100 filtration. Kinase the cDNAs (Maniatis etal. 1982) and iso late the fragments of interest after gel electrophoresis. Ligate to the blunt-cut and phosphatased vector and identify the recombinants with a colony lift procedure (Buluwela et al. 1989) by using a probe containing sequences within the amplified fragment. Directional Cloning Inactivate the residual Taq Polymerase with phenol-chloroform and ethanol precipitation. Cleave the amplified products at o n e of the adapter primer restriction sites {Xho\, Sa/I, or C/al) and at a site in the gene-specific primer or o n e known to exist in the amplified
4. RACE: Rapid Amplification of cDNA Ends
33
product. Gel purify and subclone as usual. Although a very high proportion of the recombinant plasmids may contain the desired product, it is still helpful to use colony lifts and Southern blot hy bridization (as previously described) whenever possible. It is also important to compare the size of amplified products before and after restriction to ensure that the endonucleases chosen are not cleaving within the uncharacterized portion of the cDNA. If this oc curs, try other restriction enzymes or clone the cDNAs intact.
Troubleshooting and Comments on Protocol Steps Artifactual and Truncated Products The most obvious artifact of the RACE protocol is nonspecific am plification of c D N A s that share no significant homology with the gene of interest and that fail to hybridize to an appropriate probe under Southern blot analysis conditions. Several ways of minimiz ing this problem are discussed below. It is also possible to generate specific products that are undesirable because they are truncated or because they are derived from con taminating genomic DNA. Truncation of cDNA Ends During Reverse Transcription Occa sionally, truncated c D N A 3 ' ends are produced because of annealing of the dT 1-adapter primer to an A-rich region in the transcript up 7 stream of the poly(A) tail. T h i s can be minimized by decreasing the concentration of adapter primer to 15 ng per reverse transcription reaction. For the 5' end, a primer that binds inappropriately to the message of interest within the region to be amplified will cause premature termination of the reverse transcription reaction at that site, result ing in truncated ends. Problems of this sort are minimized by (1) using short primers (12 to 16 nucleotides) rich in A + T , (2) increasing the reverse transcription temperature to 52°C, (3) decreasing the primer concentration to 15 ng or lower per reaction, and (4) trying different primers for reverse transcription. It should also be noted that primers rich in G + C produce more nonspecific reverse tran scription and thus increase background amplification.
34
Part O n e . Basic M e t h o d o l o g y
Truncation of cDNAs During Amplification Truncation of c D N A s can also occur during amplification through mismatched annealing of any of the primers. Some of these products can be detected by leaving out one primer from the reaction mixture and determining whether specific products are still amplified. More precisely, exam ine the products of an amplification reaction in which only the , gene-specific primer (for 3'-end amplifications) or the gene-specific primer and the adapter primer (for 5 -end amplifications) are present. Under these conditions, a specific product should not appear, or at least should be a different size compared to that of the amplification products generated by reactions containing all of the necessary primers. If such problems arise, mismatched annealing can often be reduced by decreasing the primer concentrations (to as little as 2.5 pmol in some cases). Production of Specific Products by Amplification of Genomic DNA It is often possible, through mismatched annealing of one of the RACE or gene-specific primers, to obtain amplification of a frag ment of the gene of interest from genomic D N A . Although reversetranscribed cDNAs should normally be present in vast excess over residual genomic DNA, technical problems with preparation of c D N A templates or with inefficient amplification through regions with secondary structure may result in preferential genomic ampli fication. Genomic D N A (50 ng) should be amplified under the same conditions as those for the c D N A pool; the results of this amplifica tion will indicate whether such products can be efficiently created and will provide information about their size. One way to minimize the problem is to use primers that span splice borders in the c D N A to force amplification of c D N A s only. A primer thus chosen should fail to anneal to the genomic DNA, since in that substrate the primer sequence is interrupted by an intron sequence. More generally, it is useful to design the amplification scheme such that the final RACE products are composed of more than one exon. This ensures that the material recovered derives from cDNAs and not from the amplification of genomic DNA.
Reverse Transcription
5
There are ~ 1 0 copies of a rare transcript in 2 0 0 ng of total RNA. Thus, very little R N A is required to provide starting material suffi-
4. RACE: Rapid Amplification of cDNA Ends
35
cient for many amplifications. If it proves difficult to obtain fully ex tended cDNAs, try using Mo-MuLV reverse transcriptase, increasing reverse transcription temperature, or pretreating the R N A with methyl mercury. For the amplification of some genes, RNase H and RNase A treatment of the c D N A - R N A hybrids after reverse tran scription appears to result in a higher yield of specific product.
Tailing An obvious advantage of tailing the first-strand c D N A s with dAs is that the same dT 1-adapter primer used in 3'-end c D N A amplifica 7 tion reactions can be used as a primer for the second-strand syn thesis of 5'-end cDNAs. In theory, any nucleotide can be used in the tailing reaction. In practice, however, it is probably best not to use either dC or dG, since this is more likely to lead to truncation of products due to nonspecific primer annealing during amplification (see "Truncation of cDNAs During Amplification") than tailing with dA or dT would. T h i s is because homopolymers of C or G an neal with much higher affinity than do homopolymers of A or T. Therefore, much shorter stretches of Cs or Gs than As or T s are re quired in a c D N A sequence for nonspecific binding of the comple mentary homopolymer-adapter primer and consequent truncation to occur during amplification.
Amplifications Nonspecific amplification is quite temperature-dependent, and the relative abundance of specific product often increases dramatically with increased annealing temperatures. Optimize the annealing temperature by repeating the amplification step at increasing tem peratures (by increments of 2°C) until specific products are no longer obtained.
Gene-Specific Primers for Amplification Nonspecific amplification will also be minimized if all of the prim ers have similar melting temperatures. Thus, choose gene-specific primers that match the adapter primer, both in length and G + C content.
36
Part O n e . Basic M e t h o d o l o g y
Controls Always include the following controls: (1) no DNA; (2) genomic DNA (see "Production of Specific Products by Amplification of Genomic DNA"); and (3) c D N A pool, amplified with gene-specific primer only (see "Truncation of cDNAs During Amplification"). Optional controls that can be included are: (1) "sham c D N A " (no specific amplification product should be obtained if reverse tran scriptase is left out when one is preparing the c D N A pool, demon strating that the putative specific product is not generated by ampli fication of tailed contaminant or residual genomic DNA) and (2) "DNased" RNA (if genomic D N A proves to be responsible for a sig nificant fraction of the gene-specific amplification, there are several RNase-free DNases available that can be used before reverse tran scription to eliminate the genomic DNA).
Cloning When nonspecific amplification is a problem, an increase in the fre quency of the desired products can be obtained during the cloning step. First, as noted in the protocol, the specific products can be iso lated from agarose gels after Southern blot analysis, and cloned. By using a blunt cloning method, one can clone the RACE products in tact, eliminating the possibility of restriction endonuclease cleavage within the uncharacterized part of the specific product. On the other hand, blunt cloning requires fully extending and kinasing the P C R products and is inefficient. A further increase in the frequency of the desired product can be obtained by directional cloning of the RACE products after cleavage with appropriate restriction enzymes. By cleaving the RACE prod ucts with two endonucleases, each specific to a different primer, the cloning of any nonspecific amplification product that has the same primer at both ends will be precluded. Directional cloning can be used to obtain an even higher frequency of the desired product, providing that a restriction enzyme that recognizes infrequent sites can be used to cleave the specific product at a site internal to the gene-specific primer. Most products arising from nonspecific ampli fication will not contain this recognition site and thus will not read ily clone. Directional cloning also dictates the orientation of the in sert; this can be useful for mapping and sequencing purposes. T h e major limitation of directional cloning is that cleavage of restriction sites within the uncharacterized portion of the RACE products can
4 . RACE: Rapid A m p l i f i c a t i o n of c D N A Ends
37
result in an unclonable product. This possibility can be minimized, as described in the "Protocol" section, but not eliminated.
5 - E n d Fidelity Extended products often end with one-to-a-few extra bases, gener ally a combination of As and Gs. T h i s is presumably because of the snapback feature of reverse transcriptase, rather than because of the PCR amplification itself. Confirm 5 ' ends with genomic sequence and primer extension/RNase protection analysis.
Construction of Full-Length cDNAs Ultimately, a full-length c D N A is often sought. T h e overlapping 3 ' and 5'-end RACE products can be linked by several approaches. First, any unique restriction site in the region of overlap can be used to join the two ends in a standard subcloning procedure. Second, the rapid mutagenesis procedure (Chapter 22) can be used at any posi tion within the overlapping region to join the two ends. Finally, in formation obtained from the RACE protocol allows one to make primers that represent the sequences at the extreme 3 ' and 5 ' ends of the m R N A . T h e s e primers can then be used to amplify full-length cDNAs from the original c D N A pool. This approach has several ad vantages. First, it minimizes the overall number of amplification cycles, thus decreasing the number of polymerase-induced muta tions. Second, this approach "strips" the c D N A of the homopolymeric sequences appended to the 5' end by the RACE protocol, which may in some cases inhibit translation. Finally, in the case of multiple 3 ' or 5' ends, successful production of a full-length c D N A confirms that the sequences used for amplification represent bona fide ends of an m R N A present in the original population.
Literature Cited Buluwela, L., A. Forster, T. Boehm, and T. H. Rabbitts. 1989. A rapid procedure for colony screening using nylon filters. Nucleic Acids Res. 1 7 : 4 5 2 . Coleclough, C. 1987. Use of primer-restriction end adapters in c D N A cloning. Meth ods Enzymol. 1 5 9 : 6 4 - 8 3 . Efstratiadis, A., F. C. Kafotos, and T. Maniatis. 1977. T h e primary structure of rabbit /3-globin m R N A as determined from cloned DNA. Cell 1 0 : 5 7 1 - 5 8 6 . Frohman, M. A., M. K. Dush, and G. R. Martin. 1988. Rapid production of full-length
38
Part O n e . Basic M e t h o d o l o g y
cDNAs from rare transcripts: amplification using a single gene-specific oligo nucleotide primer. Proc. Natl. Acad. Sci. USA 8 5 : 8 9 9 8 - 9 0 0 2 . Gubler, U., and B. J. Hoffman. 1983. A simple and very efficient method for generating c D N A libraries. Gene 2 5 : 2 6 3 - 2 6 9 . Heidecker, G., and }. Messing. 1987. A method for cloning full-length c D N A in plasmid vectors. Methods Enzymol. 1 5 9 : 2 8 - 4 1 . Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. In Molecular cloning: A laboratory manual, p. 123. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Okayama, H., M. Kawaichi, M. Brownstein, F. Lee, T. Yokota, and K. Arai. 1987. Highefficiency cloning of full-length cDNA: construction and screening of c D N A ex pression libraries for mammalian cells. Methods Enzymol. 1 5 9 : 3 - 2 7 .
Although the technology of c D N A cloning has been steadily grow ing more powerful (Gubler and Hoffman 1983; Heidecker and Mes sing 1987; Efstratiadis et al 1977} Okayama et al 1987; Coleclough 1988), the generation of full-length c D N A copies of m R N A tran scripts is often challenging, particularly with respect to obtain ing the 5' ends of messages. T h e first step, reverse transcription of mRNA, is usually the limiting one, and many variations have been described, including the use of different types of reverse transcrip tase, pretreatment of the m R N A to decrease secondary structure, al teration of reverse transcriptase conditions, and the use of primerextended c D N A instead of oligo(dT)-primed reverse transcription. Such modifications can profoundly affect the likelihood of obtaining full-length c D N A clones, but optimal conditions for reverse tran scription of a given m R N A are rarely established, since weeks to months are required to prepare and screen a single c D N A library and to isolate and analyze candidate cDNAs. The method described here, known as the Rapid Amplification of c D N A Ends (RACE) protocol (Frohman et al 1988), differs markedly in that the analysis of the reverse transcribed products can be per formed within 1 to 2 days of the start of the experiment. T h e m R N A 28
PCJR Protocols: A Guide to Methods and Applications Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
4 . RACE: Rapid Amplification of c D N A Ends
29
reverse transcription step can be modified and repeated until it is clear that the desired c D N A has been produced. Furthermore, infor mation about alternate splicing and promoter use is obtained, and the RACE protocol can be used to separate c D N A s produced from different transcripts prior to cloning steps. Finally, RACE products can be efficiently cloned into standard high-copy plasmid vectors, allowing many independent isolates to be obtained from a single plate of transformants. In essence, the RACE protocol generates cDNAs by using P C R to amplify copies of the region between a single point in the transcript and the 3 ' or 5' end. To use the RACE protocol, one must know a short stretch of sequence from an exon. From this region, primers oriented in the 3 ' and 5 ' directions are chosen that will produce overlapping c D N A s when fully extended. These primers provide specificity to the amplification step. Extension of the c D N A s from the ends of the messages to the specific primer sequences is accom plished by using primers that anneal to the natural (3' end) or a syn thetic (5' end) poly(A) tail. Finally, the overlapping 3 ' - and 5'-end RACE products are combined to produce an intact full-length c D N A . Figure 1 illustrates this strategy in more detail. In brief, for the 3 ' end, m R N A is reverse transcribed using a "hybrid" primer consist ing of oligo(dT) (17 residues) linked to a unique 17-base oligonucleo tide ("adapter") primer. Amplification is subsequently performed using the adapter primer, which binds to each c D N A at its 3 ' end, and a primer specific to the gene of interest. For the 5 ' end, reverse transcription is performed using a gene-specific primer. A homopolymer is then appended by using terminal transferase to tail the first-strand reaction products. Finally, amplification is accom plished using the hybrid primer previously described and a second gene-specific primer upstream of the first one. Additional factors considered in the development of the RACE protocol are described elsewhere (Frohman et al. 1988).
RACE Protocol
Materials
+
RNA: 1 f i g of poly(A) RNA is ideal; o n e can use as little as 200 ng of total RNA without modifying the procedure.
Part O n e . Basic M e t h o d o l o g y
30
RACE:
3' end
5' end
RACE:
mRNA VWVWWWVWvVM/WVWW <{2
cDNA
(-)
cDNA
7
I denature anneal
STRAND
« + j ( - ) STRAND
|5RTl
I remove excess 5RT primer
primer[3 « m p |
Uil cDNAs with dATP T
|3'»mp|
( • ) STRAND
t~> 8TRAN»
AAAAAAAARAI
r »
ITTTT**»*I
7
denature anneal primer fr***! xtend
STRAND gttlf i denature anneal primer |****TTTTl extend T
( +)
,TTTT| AAAAAAAAAAI
|?'»mpl
; (TO ^ R A N D "xls
(4> STRANP
TR
40 cycles
STRAND
denature anneal primer |5'»mp|
( - ) STRAND \*T*
I denature I anneal primers: I3'»n»pl and extend
( +) ^
STRAND
T R ( - ) STRAND | 5 ' . m p |
up to 6 • 10
|3+«n»pl copies
TR
7
denature anneal primer |»***j extend
(+) STRAND" £ - ) STRAND
of cDNA T R ( + ) STRAND I
}-¥•
T R C - } STRAND
Primers: ****
T -adapter: T _ dX 1X7 X
5'
I denature I anneal primers: \****\ and | S ' i m p |
GACTCGAGTC-
GACATCGATTTTTTTTTTTTTTTTT 3' T h i s sequence Clal
c o n t a i n s t h e Xho\,
Sail,
and
extend
recognition sites.
•
a
* * * * _ d a p t e r : 5' GACTCGAGTCGACATCG 3 ' 3'
4 0 cycles
up to
A M P — s p e c i f i c t o gene of interest, c o m p l e F***4
m e n t a r y t o ( - ) strand. 5RT
and 5 ' A M P — s p e c i f i c t o gene of interest,
I
T R ( » ) STRANP
I
6
T R < ~ ) STRAND |5TS71
- 10
Figure 1
S c h e m a t i c r e p r e s e n t a t i o n o ft h e RACE p r o t o c o l . O p e n r e c t a n g l e s
D N A strands actively b e i n g synthesized; shaded rectangles represent D N A synthesized. A t each
step t h ediagram
copies
of cDNA
c o m p l e m e n t a r y t o ( + ) strand.
represent previously
is s i m p l i f i e d t o i l l u s t r a t e o n l y h o w
the n e w
p r o d u c t f o r m e d d u r i n g t h e p r e v i o u s s t e p is u t i l i z e d . A ( - ) o r ( + ) s t r a n d is d e s i g n a t e d as " t r u n c a t e d " (TR) w h e n i t is s h o r t e r t h a n t h e o r i g i n a l ( - ) o r ( + ) s t r a n d , r e s p e c t i v e l y .
1 0 x RTC buffer: 500 mMTris-HCI (pH8.15) at41°C, 60 mMMgCI 2, 400 mM KCI, 10 mM DTT, each dNTP at 10 mM Hybrid dT 1-adapter primer: 7 GACTCGAGTCGACATCG AI I I I I I I I I I I I I I I I I—stock (1 /xg/ml) Adapter primer: GACTCGAGTCGACATCG Gene-specific primers: see Fig. 1
4 . RACE: Rapid Amplification of cDNA Ends
31
1 0 x PCR buffer: 670 mM Tris-HCI (pH 8.8 at room temperature), 67 mM MgCI 2, 1.7 mg/ml of bovine serum albumin, 166 mM ( N H 4) 2S 0 4 TE: 10 mM Tris-HCI (pH 7.6), 1 mM ETDA.
Amplification of cDNA 3 ' Ends Reverse Transcription 1. Assemble on ice: 2 /xl 1 0 x RTC buffer, 10 units of RNasin (Promega Biotech), 0.5 /xg of dT 1-adapter primer, and 10 units of 7 AMV reverse transcriptase (Life Sciences) in a total volume of 3.5 / x l . 2. Heat RNA in 16.5 /xl of H 20 for 3 minutes at 65°C; quench on ice and add to above. 3. Incubate at 42°C for 1 hour, and then at 52°C for 30 minutes. Dilute to 1 ml with TE and store at 4°C ("cDNA pool"). Amplification 1. Prepare PCR cocktail
5 /xl 1 0 x PCR Buffer 5 /xl DMSO 5 /xl 1 0 x dNTPs (15 mM each) 30 /xl H 20 1 /xl adapter primer (25 pmol//xl) 1 /xl gene-specific primer (25 pmol//xl) 1 to 5 /xl cDNA pool
2. Denature at 95°C for 5 minutes; cool to 72°C. Add 2.5 units of Taq polymerase and overlay with 30 /xl of min eral oil (heated to 72°C). Anneal at 55°C for 5 minutes; extend at 72°C for 40 minutes. Cycle: 95°C for 40 seconds / 55°C for 1 minute / 72°C for 3 minutes—for 40 cycles. Extend 72°C for 15 minutes.
Amplification of cDNA 5 ' Ends 1. Reverse transcribe RNA as previously described, but substitute 10 pmol gene-specific primer 1 for dT 1-adapter primer. 7
32
Part One. Basic M e t h o d o l o g y
2. Remove excess primer by using a Centricon 100 spin filter (Amicon Corp.). Dilute reverse transcription mixture with 2 ml of 0.1 x TE and centrifuge at 1000 x g for 20 minutes. Repeat and collect retained liquid. Concentrate to 10 /xl using Speed Vac centrifugation. 3. Add 4 /xl of 5 x Tailing Buffer (Bethesda Research Laboratories, Inc.), 4 /xl of 1 mM dATP, and 10 units of Terminal d Transferase (BRL). Incubate 5 minutes at 37°C, and then 5 minutes at 65°C. Dilute to 500 /xl with TE. 4. Amplify 1 to 10 /xl of cDNAs as previously described, adding dT 1-adapter primer (10 pmol) in addition to the adapter primer 7 and gene-specific primer 2.
Analysis Perform a Southern blot analysis of the amplification products. Test for single-stranded DNA by removing the PCR buffer (Centricon fil ter [any type], spun column, or ethanol precipitation) and incubat ing in mung bean nuclease buffer (BRL) and 1 unit of mung bean nu clease for 30 minutes at 30°C (Frohman et al. 1988). Compare to the samples incubated in buffer alone and in TE.
Cloning RACE Products Blunt Cloning Remove the PCR buffer and excess primer by three rounds of Cen tricon 100 filtration. Kinase the cDNAs (Maniatis etal. 1982) and iso late the fragments of interest after gel electrophoresis. Ligate to the blunt-cut and phosphatased vector and identify the recombinants with a colony lift procedure (Buluwela et al. 1989) by using a probe containing sequences within the amplified fragment. Directional Cloning Inactivate the residual Taq Polymerase with phenol-chloroform and ethanol precipitation. Cleave the amplified products at o n e of the adapter primer restriction sites {Xho\, Sa/I, or C/al) and at a site in the gene-specific primer or o n e known to exist in the amplified
4. RACE: Rapid Amplification of cDNA Ends
33
product. Gel purify and subclone as usual. Although a very high proportion of the recombinant plasmids may contain the desired product, it is still helpful to use colony lifts and Southern blot hy bridization (as previously described) whenever possible. It is also important to compare the size of amplified products before and after restriction to ensure that the endonucleases chosen are not cleaving within the uncharacterized portion of the cDNA. If this oc curs, try other restriction enzymes or clone the cDNAs intact.
Troubleshooting and Comments on Protocol Steps Artifactual and Truncated Products The most obvious artifact of the RACE protocol is nonspecific am plification of c D N A s that share no significant homology with the gene of interest and that fail to hybridize to an appropriate probe under Southern blot analysis conditions. Several ways of minimiz ing this problem are discussed below. It is also possible to generate specific products that are undesirable because they are truncated or because they are derived from con taminating genomic DNA. Truncation of cDNA Ends During Reverse Transcription Occa sionally, truncated c D N A 3 ' ends are produced because of annealing of the dT 1-adapter primer to an A-rich region in the transcript up 7 stream of the poly(A) tail. T h i s can be minimized by decreasing the concentration of adapter primer to 15 ng per reverse transcription reaction. For the 5' end, a primer that binds inappropriately to the message of interest within the region to be amplified will cause premature termination of the reverse transcription reaction at that site, result ing in truncated ends. Problems of this sort are minimized by (1) using short primers (12 to 16 nucleotides) rich in A + T , (2) increasing the reverse transcription temperature to 52°C, (3) decreasing the primer concentration to 15 ng or lower per reaction, and (4) trying different primers for reverse transcription. It should also be noted that primers rich in G + C produce more nonspecific reverse tran scription and thus increase background amplification.
34
Part O n e . Basic M e t h o d o l o g y
Truncation of cDNAs During Amplification Truncation of c D N A s can also occur during amplification through mismatched annealing of any of the primers. Some of these products can be detected by leaving out one primer from the reaction mixture and determining whether specific products are still amplified. More precisely, exam ine the products of an amplification reaction in which only the , gene-specific primer (for 3'-end amplifications) or the gene-specific primer and the adapter primer (for 5 -end amplifications) are present. Under these conditions, a specific product should not appear, or at least should be a different size compared to that of the amplification products generated by reactions containing all of the necessary primers. If such problems arise, mismatched annealing can often be reduced by decreasing the primer concentrations (to as little as 2.5 pmol in some cases). Production of Specific Products by Amplification of Genomic DNA It is often possible, through mismatched annealing of one of the RACE or gene-specific primers, to obtain amplification of a frag ment of the gene of interest from genomic D N A . Although reversetranscribed cDNAs should normally be present in vast excess over residual genomic DNA, technical problems with preparation of c D N A templates or with inefficient amplification through regions with secondary structure may result in preferential genomic ampli fication. Genomic D N A (50 ng) should be amplified under the same conditions as those for the c D N A pool; the results of this amplifica tion will indicate whether such products can be efficiently created and will provide information about their size. One way to minimize the problem is to use primers that span splice borders in the c D N A to force amplification of c D N A s only. A primer thus chosen should fail to anneal to the genomic DNA, since in that substrate the primer sequence is interrupted by an intron sequence. More generally, it is useful to design the amplification scheme such that the final RACE products are composed of more than one exon. This ensures that the material recovered derives from cDNAs and not from the amplification of genomic DNA.
Reverse Transcription
5
There are ~ 1 0 copies of a rare transcript in 2 0 0 ng of total RNA. Thus, very little R N A is required to provide starting material suffi-
4. RACE: Rapid Amplification of cDNA Ends
35
cient for many amplifications. If it proves difficult to obtain fully ex tended cDNAs, try using Mo-MuLV reverse transcriptase, increasing reverse transcription temperature, or pretreating the R N A with methyl mercury. For the amplification of some genes, RNase H and RNase A treatment of the c D N A - R N A hybrids after reverse tran scription appears to result in a higher yield of specific product.
Tailing An obvious advantage of tailing the first-strand c D N A s with dAs is that the same dT 1-adapter primer used in 3'-end c D N A amplifica 7 tion reactions can be used as a primer for the second-strand syn thesis of 5'-end cDNAs. In theory, any nucleotide can be used in the tailing reaction. In practice, however, it is probably best not to use either dC or dG, since this is more likely to lead to truncation of products due to nonspecific primer annealing during amplification (see "Truncation of cDNAs During Amplification") than tailing with dA or dT would. T h i s is because homopolymers of C or G an neal with much higher affinity than do homopolymers of A or T. Therefore, much shorter stretches of Cs or Gs than As or T s are re quired in a c D N A sequence for nonspecific binding of the comple mentary homopolymer-adapter primer and consequent truncation to occur during amplification.
Amplifications Nonspecific amplification is quite temperature-dependent, and the relative abundance of specific product often increases dramatically with increased annealing temperatures. Optimize the annealing temperature by repeating the amplification step at increasing tem peratures (by increments of 2°C) until specific products are no longer obtained.
Gene-Specific Primers for Amplification Nonspecific amplification will also be minimized if all of the prim ers have similar melting temperatures. Thus, choose gene-specific primers that match the adapter primer, both in length and G + C content.
36
Part O n e . Basic M e t h o d o l o g y
Controls Always include the following controls: (1) no DNA; (2) genomic DNA (see "Production of Specific Products by Amplification of Genomic DNA"); and (3) c D N A pool, amplified with gene-specific primer only (see "Truncation of cDNAs During Amplification"). Optional controls that can be included are: (1) "sham c D N A " (no specific amplification product should be obtained if reverse tran scriptase is left out when one is preparing the c D N A pool, demon strating that the putative specific product is not generated by ampli fication of tailed contaminant or residual genomic DNA) and (2) "DNased" RNA (if genomic D N A proves to be responsible for a sig nificant fraction of the gene-specific amplification, there are several RNase-free DNases available that can be used before reverse tran scription to eliminate the genomic DNA).
Cloning When nonspecific amplification is a problem, an increase in the fre quency of the desired products can be obtained during the cloning step. First, as noted in the protocol, the specific products can be iso lated from agarose gels after Southern blot analysis, and cloned. By using a blunt cloning method, one can clone the RACE products in tact, eliminating the possibility of restriction endonuclease cleavage within the uncharacterized part of the specific product. On the other hand, blunt cloning requires fully extending and kinasing the P C R products and is inefficient. A further increase in the frequency of the desired product can be obtained by directional cloning of the RACE products after cleavage with appropriate restriction enzymes. By cleaving the RACE prod ucts with two endonucleases, each specific to a different primer, the cloning of any nonspecific amplification product that has the same primer at both ends will be precluded. Directional cloning can be used to obtain an even higher frequency of the desired product, providing that a restriction enzyme that recognizes infrequent sites can be used to cleave the specific product at a site internal to the gene-specific primer. Most products arising from nonspecific ampli fication will not contain this recognition site and thus will not read ily clone. Directional cloning also dictates the orientation of the in sert; this can be useful for mapping and sequencing purposes. T h e major limitation of directional cloning is that cleavage of restriction sites within the uncharacterized portion of the RACE products can
4 . RACE: Rapid A m p l i f i c a t i o n of c D N A Ends
37
result in an unclonable product. This possibility can be minimized, as described in the "Protocol" section, but not eliminated.
5 - E n d Fidelity Extended products often end with one-to-a-few extra bases, gener ally a combination of As and Gs. T h i s is presumably because of the snapback feature of reverse transcriptase, rather than because of the PCR amplification itself. Confirm 5 ' ends with genomic sequence and primer extension/RNase protection analysis.
Construction of Full-Length cDNAs Ultimately, a full-length c D N A is often sought. T h e overlapping 3 ' and 5'-end RACE products can be linked by several approaches. First, any unique restriction site in the region of overlap can be used to join the two ends in a standard subcloning procedure. Second, the rapid mutagenesis procedure (Chapter 22) can be used at any posi tion within the overlapping region to join the two ends. Finally, in formation obtained from the RACE protocol allows one to make primers that represent the sequences at the extreme 3 ' and 5 ' ends of the m R N A . T h e s e primers can then be used to amplify full-length cDNAs from the original c D N A pool. This approach has several ad vantages. First, it minimizes the overall number of amplification cycles, thus decreasing the number of polymerase-induced muta tions. Second, this approach "strips" the c D N A of the homopolymeric sequences appended to the 5' end by the RACE protocol, which may in some cases inhibit translation. Finally, in the case of multiple 3 ' or 5' ends, successful production of a full-length c D N A confirms that the sequences used for amplification represent bona fide ends of an m R N A present in the original population.
Literature Cited Buluwela, L., A. Forster, T. Boehm, and T. H. Rabbitts. 1989. A rapid procedure for colony screening using nylon filters. Nucleic Acids Res. 1 7 : 4 5 2 . Coleclough, C. 1987. Use of primer-restriction end adapters in c D N A cloning. Meth ods Enzymol. 1 5 9 : 6 4 - 8 3 . Efstratiadis, A., F. C. Kafotos, and T. Maniatis. 1977. T h e primary structure of rabbit /3-globin m R N A as determined from cloned DNA. Cell 1 0 : 5 7 1 - 5 8 6 . Frohman, M. A., M. K. Dush, and G. R. Martin. 1988. Rapid production of full-length
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Part O n e . Basic M e t h o d o l o g y
cDNAs from rare transcripts: amplification using a single gene-specific oligo nucleotide primer. Proc. Natl. Acad. Sci. USA 8 5 : 8 9 9 8 - 9 0 0 2 . Gubler, U., and B. J. Hoffman. 1983. A simple and very efficient method for generating c D N A libraries. Gene 2 5 : 2 6 3 - 2 6 9 . Heidecker, G., and }. Messing. 1987. A method for cloning full-length c D N A in plasmid vectors. Methods Enzymol. 1 5 9 : 2 8 - 4 1 . Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. In Molecular cloning: A laboratory manual, p. 123. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Okayama, H., M. Kawaichi, M. Brownstein, F. Lee, T. Yokota, and K. Arai. 1987. Highefficiency cloning of full-length cDNA: construction and screening of c D N A ex pression libraries for mammalian cells. Methods Enzymol. 1 5 9 : 3 - 2 7 .