Molecular Techniques and Methods

5'-RACE
(Rapid Amplification of 5'-cDNA Ends)

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Most attempts to identify and isolate a novel cDNA result in the acquisition of clones that represent only a part of the mRNA's complete sequence. The missing sequence (cDNA ends) can be cloned by PCR, using a technique called rapid amplification of cDNA ends (RACE). RACE cloning is advantageous for several reasons. It takes weeks to screen cDNA libraries, obtain individual cDNA clones, and analyze the clones to determine if the missing sequence is present; using PCR, such information can be generated within a few days. As a result, it becomes practical to modify RNA preparation and/or reverse transcription conditions until full-length cDNAs are generated and observed.

In RACE technique, PCR is used to amplify partial cDNAs representing the region between a single point in a mRNA transcript and its 3'- or 5'-end. A short internal stretch of sequence must already be known from the mRNA of interest. From this sequence, gene-specific primers are chosen that are oriented in the direction of the missing sequence. Extension of the partial cDNAs from the unknown end of the message back to the known region is achieved using primers that anneal to the preexisting poly(A) tail (3'-end) or to an appended homopolymer tail (5'-end). Using RACE, enrichments on the order of 106- to 107-fold can be obtained. To generate "5'-end" partial cDNA clones, reverse transcription (primer extension) is carried out using a gene-specific primer to generate first-strand products. Then, a Poly(A) tail is appended using terminal deoxynucleotidyltransferase (TdT) and dATP. Amplification is then achieved to form the second strand of cDNA. Finally, a second set of PCR cycles is carried out using nested primers to increase specificity.






MATERIALS AND SOLUTIONS

The materials required can be purchased from most major suppliers.


5 x Reverse Transcription Buffer (10 ml)
250 mM Tris-HCl (pH 8.3) ----------------------- 2.5 ml of 1 M Tris-HCl
375 mM KCl ------------------------------------- 3.75 ml of 1 M KCl
15 mM MgCl2 ------------------------------------ 0.15 ml of 1 M MgCl2
Distilled H2O -------------------------------------- 3.6 ml


5 x Tailing Buffer (10 ml)
125 mM Tris-HCl (pH 6.6) ------------------------ 1.25 ml of 1 M Tris-HCl
1 M Potassium cacodylate ------------------------- 1.8 g
1250 ug/ml BSA ---------------------------------- 12.5 mg
Add Distilled H2O to make a final volume of------ 3.6 ml




PROCEDURES

First-Strand cDNA Synthesis by Reverse Transcription

1. Do reverse transcription by assembling the following components on ice.

5 x Reverse Transcription Buffer ------------------------ 4 ul
dNTPs (10 mM of each dNTP) -------------------------1 ul
0.1 M DTT --------------------------------------------- 2 ul
RNasin ------------------------------------------------- 0.5 ul

2. Mix the following components together in an another microfuge tube.

3'-Primer (100 ug/ ul) ----------------------------------- 500 ul
Poly A+ RNA ------------------------------------------- 1 ug
(or, Total RNA ------------------------------------------ 5 ug)
Add DEPC-treated H2O to make a final volume of ----- 13 ul

  • Heat for 3 minutes at 80oC and cool rapidly on ice.
  • Spin for 5 seconds in a microfuge.
  • Add to the reverse transcription components from step 1.

    3. Add 1 ul (200 units) of SuperScript II reverse transcriptase, and incubate for 1 hour at 42oC, and 10 minutes at 50oC.

    4. Incubate for 15 minutes at 70oC to inactivate reverse transcriptase.
  • Spin for 5 seconds in a microfuge.

    5. Add 1 ul (1-2 units) of RNase H to the tube and incubate for 20 minutes at 37oC to destroy the RNA template.

    6. Dilute the reaction mixture to 400 ul with TE and store at 4oC.
  • (This is 5'-end non-tailed cDNA pool).



    Appending a Poly A+-Tail to First-Strand cDNA

  • The conditions described below result in the addition of 30-400 nucleotides.

    7. Remove excess primer using spin columns (ex. MICROCON-100 spin filters). The final volume recovered should not exceed 10 ul. Adjust volume to 10 ul using distilled H2O.

    8. Add the following components.

    5 x Tailing Buffer -------------------- 4 ul
    1.5 mM CoCl2 ---------------------- 1.2 ul of 25 mM CoCl2
    1 mM dATP ------------------------ 4 ul
    TdT (Terminal transferase) ---------- 10 units

    9. Incubate for 5 minutes at 37oC and then 5 minutes at 65oC.

    10. Dilute to 500 ul with TE.
  • (This is 5'-end tailed cDNA pool).



    First Round PCR Amplification

    11. Mix the following components in a PCR tube.

    5'-end tailed cDNA pool from step 10
    1 ul
    25 pmole 3'-Primer
    2.5 ul of 500 pmole 3'-Primer
    25 pmole Oligo dT26-28-Primer for 5'-region
    2.5 ul of 500 pmole Oligo dT26-28
    5 x Taq DNA Polymerase Buffer
    10 ul
    1.5 mM dATP
    1.5 ul of 50 mM dATP
    1.5 mM dCTP
    1.5 ul of 50 mM dCTP
    1.5 mM dGTP
    1.5 ul of 50 mM dGTP
    1.5 mM dTTP
    1.5 ul of 50 mM dTTP
    10% DMSO
    5 ul of 100% DMSO
    Add distilled H2O to make a final volume of
    50 ul


    12. Heat in a thermal cycler for 5 minutes at 98oC to denature the first-strand products.
  • Cool to 75oC.
  • Add 2.5 units of Taq DNA polymerase.
  • Incubate for 2 minutes at the appropriate annealing temperature (48-52oC).
  • Extend the cDNAs for 40 minutes at 72oC.

    13. Carry out 30 cycles of PCR amplification using a step program, followed by a 15-minute final extension at 72oC.
  • Cool to room temperature.

    Cycles
    Time
    Temperature (oC)
    30
    30 sec
    95oC
    30 sec
    48-52oC
    2-3 min
    72oC
    1
    15 min
    72oC



    Second Round PCR Amplification

    14. Dilute 1 ul of the amplification products from the First Round PCR (from step 13) into 20 ul of TE.

    15. Mix the following components in a PCR tube.

    20 x Diluted First Round PCR products from step 14
    1 ul
    25 pmole 3'-nested Primer
    2.5 ul of 500 pmole 3'-nested Primer
    25 pmole Oligo dT26-28-Primer for 5'-region
    2.5 ul of 500 pmole Oligo dT26-28
    5 x Taq DNA Polymerase Buffer
    10 ul
    1.5 mM dATP
    1.5 ul of 50 mM dATP
    1.5 mM dCTP
    1.5 ul of 50 mM dCTP
    1.5 mM dGTP
    1.5 ul of 50 mM dGTP
    1.5 mM dTTP
    1.5 ul of 50 mM dTTP
    10% DMSO
    5 ul of 100% DMSO
    Add distilled H2O to make a final volume of
    50 ul


    16. Carry out 30 cycles of PCR amplification using a step program, followed by a 15-minute final extension at 72oC.
  • Cool to room temperature.

    Cycles
    Time
    Temperature (oC)
    30
    30 sec
    95oC
    30 sec
    52-60oC
    2-3 min
    72oC
    1
    15 min
    72oC



    Cloning

    17. To clone the cDNA ends directly from the amplification reaction (or after gel purification, which is recommended), ligate an aliquot of the products to plasmid vector encoding a 1-nucleotide 3'-overhang consisting of a T on both strands. Such vector DNA is available commercially (TA Kit).



    Sequencing

    18. RACE products can be sequenced directly on a population level using a variety of protocols, including cycle sequencing, from the end at which the gene-specific primers are located.



    Hybridization Probes

    19. RACE products are generally pure enough that they can be used as probes for RNA and DNA blot analyses. It should be kept in mind that small amounts of contaminating nonspecific cDNAs are always present.




    NOTES

  • Possible Problems in Reverse Transcription

    Damaged RNA Electrophorese RNA in a 1% formaldehyde minigel and examine the integrity of the 18S and 28S ribosomal bands. Discard the RNA preparation if the ribosomal bands are not sharp.
    Contaminants Ensure that the RNA preparation is free of agents that inhibit reverse transcription, e.g., LiCl.and SDS.
    Bad Reagents To monitor reverse transcription of the RNA, add 20 uCi of [32P]dCTP to the reaction, separate newly created cDNAs using gel electrophoresis, wrap the gel in plastic wrap, and expose it to X-ray film. Accurate estimates of cDNA size can best be determined using alkaline agarose gels, but a simple 1% agarose minigel will suffice to confirm that reverse transcription took place and that cDNAs of reasonable length were generated.



  • Possible Problems in Tailing

    Bad Reagents Tail 100 ng of a DNA fragment of approximately 100-300 bp in length for 30 minutes. In addition, mock-tail the same fragment (add everything but the TdT). Run both samples in a 1% agarose minigel. The mock-tailed fragment should run as a tight band. The tailed fragment should have increased in size by 20-200 bp and should appear to run as a diffuse band that trails off into higher-molecular-weight products. If this is not observed, replace reagents.



  • Possible Problems with PCR Amplification

    No Product If no products are observed for the first set of amplifications after 30 cycles, add fresh Taq DNA polymerase and carry out an additional 15 rounds of amplification (extra enzyme is not necessary if the entire set of 45 cycles is carried out without interruption at cycle 30). Product is always observed after a total of 45 cycles if efficient amplification is taking place. If no product is observed, carry out a PCR using control templates and primers to ensure the integrity of the reagents.
    Smeared Products This is caused by too many cycles or too much starting material.

    Polymerase pausing may have occurred during the reverse transcription step. To obtain nearly full-length cDNA ends, the amplification mixture should be electrophoresed and the longest products recovered by gel isolation. An aliquot of this material can then be reamplified for a limited number of cycles.
    Nonspecific Amplification Check the sequences of cDNA and primers. If all are correct, examine primers (using a computer program) for secondary structure and self-annealing problems. Consider ordering new primers. Alternatively, secondary structure in the template may be blocking amplification. Consider adding formamide or 7aza-GTP (in a 1:3 ratio with dGTP) to the reaction to assist polymerization. 7aza-GTP can also be added to the reverse transcription reaction.

    Raise annealing temperatures gradually and sequentially in each stage of the procedure until nonspecific products are no longer observed.
    The last few base pairs
    of the 5'-end sequence do not
    match the corresponding genomic sequence.    		 Be aware that reverse transcriptase and T7 and T3 RNA polymerase can add on a few extra template-independent nucleotides.




    KIT INFORMATION




    REFERENCES

  • Borson ND, WL Salo, LR Drewes (1992) A lock-docking oligo(dT) primer for 5'- and 3'-RACE PCR. PCR Methods Appl. 2: 144-148.

  • Frohman MA (1993) Rapid amplification of cDNA for generation of full-length cDNA ends: Thermal RACE. Methods Enzymot. 218: 340-356.

  • Frohman MA, MK Dush, GR Martin (1988) Rapid production of full-length cDNAs from rare transcripts by amplification using a single gene-specific oligonucleotide primer. PNAS 85: 8098-9002.

  • Holton TA, MW Graham (1991) A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. Nucleic Acids Res. 19: 1156.

  • Kovalic D, JH Kwak, B Weisblum (1991) General method for direct cloning of DNA fragments generated by the polymerase chain reaction. Nucleic Acids Res. 19: 4650.

  • Marchuk D, M Drumm, A Saulino, FS Collins (1991) Construction of T-vector, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 19: 1154.

  • Sarker, G., S. Kapeiner, and S.S. Sommer (1990) Formamide can dramatically improve the specificity of PCR. Nucleic Acids Res. IS: 7465.

  • Schuster DM, GW Buchman, A Rastchian (1992) A simple and efficient method for amplification of cDNA ends using 5'-RACE. Focus 14: 46-52.

  • Templeton NS, E Ureelay, B Safer (1993) Reducing artifact and increasing the yield of specific DNA target fragments during PCR-RACE or anchor PCR. BioTechniques 15: 48-50.

  • Tessier DC, R Brousseau, T Vernel (1986) Ligation of single-stranded oligodeoxyribonucleotides by T4 RNA ligase. Anal. Biochem. 158: 171-178.


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