by Richard E. Manrow, Ph.D., NCI, and Lance Liotta, M.D., Ph.D., NCI
The ability to rapidly amplify cDNA ends (RACE) has accelerated the pace at which complete cDNA sequences can be obtained. This method has greatly facilitated protein sequence determination and mRNA structure, sequence, and expression studies. Useful information can now be obtained quickly, even from low-abundance mRNAs. Currently, investigators can analyze messages in samples of fewer than 100 cells; in the future, detailed analyses may be possible for the study of individual cells.
The Method and How it Works
Complete cDNA and protein sequence information is essential for structural
and expression studies and, ultimately, for the isolation and characterization of genomic clones. Unfortunately, the cDNAs obtained from most libraries are incomplete. Most frequently, they lack sequences found at the extreme 5' ends of their mRNA templates. The principal reason for this is that reverse transcriptases (RTs), used to create cDNAs from mRNA templates, often fail to traverse entire mRNA molecules during cDNA synthesis. Until the development of 5' RACE technology, investigators had no choice but to re-screen libraries to obtain other cDNA clones that contained the missing information. These re-screening efforts were tedious and not always successful.
The solution to this problem was simple and elegant: existing methods for adding known sequences to the 3' ends of single-stranded DNAs were coupled with the polymerase chain reaction (PCR) to selectively amplify specific cDNA targets. As is often the case when scientists are trying to solve a critical problem, more than one approach was explored. The two 5' RACE strategies that emerged (1-3) are outlined in Figure 1. In both cases, the first step involves cDNA synthesis using an oligonucleotide primer (G1) that anneals to the known mRNA template for the sequences to be extended. Most investigators employ either avian myeloblastosis virus (AMV) RT or Moloney murine leukemia virus (MMLV) RT to accomplish this. However, a DNA polymerase from the thermophilic bacterium Thermus thermophilus has recently been shown to exhibit a strong RT activity in the presence of Mn+2 (4); use of this enzyme (Tth DNA polymerase) at elevated temperatures (i.e., 70 deg.C) may enhance the likelihood that cDNA synthesis will not terminate prematurely on GC-rich templates or at sites possessing secondary structure (5).
The two 5' RACE strategies diverge at the next step, in which specific DNA sequences are linked to the 3' ends of the newly synthesized cDNAs in order to mark the new, extended sequences for amplification. In one approach, homopolymeric tails are added using the enzyme terminal deoxynucleotidyl transferase (terminal transferase, or TdT). Subsequently, PCR amplification of the tailed cDNA is performed using a second gene-specific primer (G2; nested with respect to the cDNA primer) and a mixture of two other primers, each bearing the same unique sequence (an arbitrary "anchor" sequence determined by the investigator) and one of them bearing, in addition, a homopolymeric tail complementary to the tails added to the cDNA. In this way, the sequence of interest becomes flanked by unique sequences (i.e., a known cDNA sequence and the anchor sequence), thereby marking it as a target for selective amplification. Use of anchor sequences in the upstream primers is important because homopolymeric primers may anneal nonspecifically to sequences other than the desired ones during PCR. Use of nested, gene-specific primers in the amplification phase also serves to increase specificity.
In the second approach, an anchor oligomer is ligated directly onto the
3' ends of the newly synthesized cDNAs using T4 RNA ligase. This approach takes advantage of observations made by Tessier et al. (6) that T4 RNA ligase will ligate two single-stranded DNA segments in the presence of hexamine cobalt chloride. Even under optimum conditions, this ligation reaction does not go to completion, but efficiencies of 40 - 60% have been reported. Given the amplifying power of PCR, even limited anchor oligomer ligation should be sufficient; however, the following precautions must be taken to ensure that the desired ligation reaction is favored and that self-ligation of the anchor oligomer or the cDNA is minimized: 1) the cDNA primer cannot have a phosphate group at its 5' end; 2) the anchor oligomer must be phosphorylated at its 5' end by using T4 polynucleotide kinase; and 3) the 3' end of the anchor oligomer must be blocked by the addition of a ddAMP moiety (or a methyl group).
The reactions unique to each of these two 5' RACE strategies work sufficiently well that commercial kits have been built around them. The 5' RACE System Kit sold by Life Technologies, Inc. (GIBCO-BRL), uses a variation of the homo-polymeric tailing method; the 5'-ampliFINDER RACE Kit from Clontech Laboratories, Inc., employs the anchor oligomer ligation method. Because some scientists dislike using kits, detailed protocols for the described reactions are provided below. Please note that these protocols and those found in the kits may differ in some aspects. Mention of specific products does not constitute an endorsement.
Protocols
Single-Stranded cDNA Synthesis and Purification
cDNA synthesis is usually performed using 1 - 2 ug poly(A)+ RNA as the template; however, successful 5' RACE has been achieved with less than 1 ng total cellular RNA (3). The G1 oligonucleotide used to prime cDNA synthesis should be approximately 20 residues long and have a GC content of 45 - 65%. Use sterile, RNAse-free water to resuspend and dilute the G1 oligomer; the other reaction components should also be RNAse-free. Typically, reaction mixtures range in volume from 20 to 40 uL. Prior to setting up the final mixture, the RNA template, the G1 primer, and the aqueous component of the mixture should
be combined in a sterile tube, heated at 65 deg.C for 5 min., and then chilled
on ice.
The reaction conditions using AMV RT are
< 1 - 2 ug RNA template
10 pmol G1 primer
50 mM Tris-HCl, pH 8.3
50 mM KCl
10 mM MgCl2
1 mM dNTPs (Na+)
1 mM DTT
1 mM EDTA
4 mM sodium pyrophosphate
10 ug/mL bovine serum albumin (BSA)
40 units placental RNAse inhibitor (RNAsin; Promega Corporation)
10 units AMV RT
Incubate at 42 deg.C for 1 hour, followed by an optional 30 min. incubation at 52 deg.C.
The reaction conditions using MMLV RT are
< 1-2 ug RNA template
10 pmol G1 primer
50 mM Tris-HCl, pH 8.3
75 mM KCl
10 mM DTT
3 mM MgCl2
0.5 mM dNTPs (Na+)
100 ug/mL BSA
40 units RNAsin
200 units MMLV RT
Incubate at 42 deg.C for 30 min..
The reaction conditions using Tth DNA polymerase RT are
< 1-2 ug RNA template
10 pmol G1 primer
10 mM Tris-HCl, pH 8.3
90 mM KCl
1 mM MnCl2 (optimization may be necessary)
0.2 mM dNTPs
5 Units Tth polymerase (available from Perkin Elmer or Epicentre Technologies)
Overlay the reaction mix with mineral oil to prevent evaporation and incubate at 70 deg.C for 15 min..
The cDNA synthesis reactions are terminated by adding EDTA to a final concentration of 15 mM (for AMV RT and MMLV RT) or by adding EGTA to a concentration of 0.75 mM (for Tth DNA polymerase). The RNA template is hydrolyzed (and the enzymes denatured) by adding NaOH to a concentration of 400 mM and incubating the mixture at 65 deg.C for 30 min. Acetic acid is then added to a concentration of 400 mM to neutralize the solution. Some investigators terminate MMLV RT reactions and simultaneously inactivate the enzyme by heating the reaction mix at 65 - 70 deg.C; they then hydrolyze the RNA template by treatment with Escherichia coli RNAse H (2 units per reaction tube). The cDNA is seperated from unused G1 oligomers and residual RNA by differential binding to a silica matrix (GENECLEAN, Bio101 Inc., or similar matrices included in the 5' RACE kits described above) in the presence of 4.0 - 4.5 M NaI. The bound cDNA is washed as recommended by the vendors and eluted with sterile, distilled water. Extreme care must be taken to avoid silica contamination of the eluted material. The volume of the cDNA sample should be adjusted to approximately 10 ul. Other approaches have been used to deal with residual G1 oligomers, ranging from doing nothing at all to removing them either by size exclusion chromatography or gel electrophoresis. The presence of unused G1 oligomers may interfere with subsequent amplification reactions.
Homopolymeric cDNA Tailing
The sample of cDNA should be heated at 90 deg.C for 2 min. to remove secondary structure and then chilled on ice. All
or part of the cDNA may be tailed. Tailing reactions are usually performed at 37 deg.C for 5 min. in 20 - 25 uL reaction mixtures containing
cDNA
100 mM potassium cacodylate, pH 7.2
2 mM CoCl2
0.2 mM DTT
0.2 mM dATP (or dCTP)
10 units TdT.
The tailing reaction is terminated by heating the mixture at 70 deg.C for 5 - 10 min. At this point, the tailed cDNA may be diluted ~25-fold with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), or it may be precipitated with ethanol, using 20 ug
of glycogen as the carrier. If the cDNA is precipitated, resuspend it in approximately 500 uL sterile, distilled water. Typically, 1 - 2% of the tailed cDNA is used in amplification reactions.
5' End Phosphorylation of an Anchor Oligomer
Prepare a 40-uL reaction mixture containing
2 nmol anchor oligomer (usually 35 - 45 nucleotides long; sequence determined by the investigator or kit manufacturer)
70 mM Tris-HCl, pH 7.5
10 mM MgCl2
1 mM DTT
0.5 mM ATP
20 Units T4 polynucleotide kinase.
Allow the phosphorylation reaction to proceed at 37 deg.C for 60 min., and then heat-inactivate the kinase by incubating the reaction tube in a 65 - 70 deg.C water bath for 20 min. Precipitate the phosphorylated oligomer with ethanol, resuspend it in sterile, distilled water (up to 50 uL), and block the 3' end of some or all of it with ddATP and TdT as described below.
3' End Blocking of an Anchor Oligomer with ddATP
A 70 ul reaction mixture is prepared containing
1 nmol anchor oligomer
0.2 mM ddATP
100 mM potassium cacodylate, pH 7.2
2 mM CoCl2
0.2 mM DTT
10 Units TdT.
The mixture is incubated at 37 deg.C for 60 min. The reaction is terminated by adding 210 uL of ice-cold 20 mM EDTA, and the blocked oligomer is precipitated with ethanol and resuspended in sterile, distilled water.
Anchor Oligomer Ligation to cDNA
All or part of the cDNA prepared above may be ligated to the anchor oligomer. Since the final volume of the ligation mixture is 10 uL, the cDNA sample may have to be concentrated by vacuum drying. The ligation reaction is performed at room temperature for 12 - 24 h. with the following components:
cDNA sample prepared above
1-10 pmol 5' phosphorylated, 3' blocked anchor oligomer
50 mM Tris-HCl, pH 8.0
10 mM MgCl2
1 mM hexamine cobalt chloride
20 umol ATP
10 ug/mL BSA
25% (w/v) PEG 8000
10 units T4 RNA ligase.
Termination of the ligation reaction is achieved by diluting the reaction mixture 1:10 with sterile, distilled water. In most cases, 1 - 5% of the diluted mixture is used in subsequent amplification reactions.
cDNA Amplification
Regardless of the strategy employed, "hot start" PCR using 50 uL reaction volumes is recommended. Use 10 pmol each of the anchor primer and the G2 or the G3 primer with cDNAs modified by the anchor oligomer ligation method; use 10 pmol of the tailed anchor primer and 25 - 100 pmol each of the anchor primer and the G2 or G3 primer with cDNAs modified by the tailing method. (Note: The 5' RACE System kit does not use a mixture of tailed and non-tailed anchor primers as the upstream oligomers; instead, only an anchor primer with a modified homopolymeric tail is used.) The following cycling parameters should yield good results:
94deg.C, 45 s.
55deg.C, 45 s.
72deg.C, 2 min.
for 35 cycles.
Troubleshooting Tips
1. Intact mRNA is absolutely essential for the success of 5' RACE. If possible, a northern blot of the starting RNA should be made to verify its integrity.
2. The cDNA primer should be designed to anneal at least 200 bases downstream from the 5' end of the known mRNA sequence. The resulting cDNA will, therefore, contain a stretch of known sequence. Amplification reactions using oligomers complementary to the ends of this region will allow confirmation that the correct target sequence has been copied. A necessary control here is the amplification of mock cDNA (i.e., from a reaction in which RT was omitted); this will establish that the final product was generated from mRNA and not from contaminating genomic DNA.
3. If cDNA yields in the first step are low, purification using a silica matrix may be risky. In this case, purify the cDNA by size-exclusion chromatography or by gel electrophoresis. If the cDNA is purified electrophoretically, it will have to be labeled to locate its position in a gel.
4. Incomplete cDNA synthesis, nonspecific priming by the oligomers
used in cDNA synthesis and PCR, and PCR artifacts such as primer-dimer formation may yield complex 5' RACE products. Therefore, it may be necessary to try several different gene-specific oligomers and to vary the amplification protocol to achieve a good result. Changes in the amplification conditions may include altering the oligomer annealing temperature, using a different MgCl2 concentration, using a different thermostable DNA polymerase, and adding 10% dimethyl sulfoxide (DMSO) to the PCR mixtures. If no specific product is observed after the first round of PCR, prepare a Southern blot with some of the amplified material to see whether a small amount of the desired species is present (use the known cDNA sequence as a probe). If the desired product is present, re-amplification with another nested, gene-specific primer may improve the yield.
5. Use of nested, gene-specific primers enhances the specificity of the amplification reactions. However, control reactions using only 5' or 3' primers
are recommended to establish bilateral priming of the PCR products.
6. The oligomers used in the amplification reactions should contain restriction enzyme recognition sequences at their 5' termini to facilitate cloning
of the products; the tailed anchor primer is synthesized with a homopolymeric
tail of approximately 15 residues at its 3' end.
5' RACE Contacts
1. Richard E. Manrow, NCI
496-9753
2. Lee Tiffany, NIAID
496-2877
3. X. Su, NIAID
496-4023
4. Nancy Templeton, MEGABIOS Corp.
(415)-802-0350; fax: (415)-802-0355
References
1. M.A. Frohman, M.K. Dush, and G.R. Martin. "Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer." Proc. Nat. Acad. Sci. USA 85, 8998 - 9002 (1988).
2. J. B. D. M. Edwards, J. Delort, and J. Mallet. "Oligodeoxyribonucleotide ligation to single-stranded cDNAs: a new tool for cloning 5' ends of mRNAs and for constructing cDNA libraries by in vitro amplification." Nucl. Acids Res. 19, 5227 - 32 (1991).
3. A.B. Troutt, M. G. McHeyzer-Williams, B. Pulendran, and G.J.V. Nossal. "Ligation-anchored PCR: a simple amplification technique with single-sided specificity." Proc. Nat. Acad. Sci. USA 89, 9823 - 25 (1992)
4. T.W. Myers and D.H. Gelfand. "Reverse transcription and DNA amplification by a Thermus thermophilus DNA Polymerase." Biochemistry 30, 7661 - 66 (1991).
5. N.S. Templeton, E. Urcelay, and B. Safer. "Reducing artifact and increasing the yield of specific target fragments during PCR-RACE or anchor PCR." Biotechniques 15, 48 - 50 (1993).
6. D.C. Tessier, R. Brousseau, and T. Vernet. "Ligation of single-stranded oligo-deoxyribonucleotides by T4 RNA ligase." Anal. Biochem. 158, 171 - 78 (1986).