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MOLECULAR BIOLOGY: WORKING WITH DNA

POLMERASE CHAIN REACTION

3' RAPID AMPLIFICATION OF cDNA ENDS (RACE) BY PCR

3' Rapid Amplification of cDNA Ends (RACE) by PCR Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

This protocol describes 3'-end amplification of mRNA by PCR. The first-strand cDNA is synthesized from total or poly(A+) RNA by priming from the poly A tail of the mRNA using a oligo (dT) adaptor primer. The cDNA is then amplified via PCR using a gene-specific primer and an adaptor primer.

Procedure Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

A. Synthesis of First Strands of cDNAs Using a (dT)-Adaptor Primer 1. Dissolve 1 to 5 μg of poly (A)+ RNA or 10 to 20 μg of total RNA in 10 μl of DEPC-treated water (see Hint #2). 2. Incubate the RNA at 65°C for 5 min. 3. Chill the RNA on ice. 4. Add the following to the RNA:    2 μl of 10X PCR Buffer    2 μl of 10 mM dNTPs    2 μl of 10 mM DTT    0.5 μl of RNasin    3 μl of 100 ng/ml (dT)17-Adaptor    1 μl of AMV Reverse Transcriptase (see Hint #3) 5. Incubate the reaction at 42°C for 1 to 2 hr. 6. Incubate the reaction at 95°C for 5 min to stop the reaction. 7. Dilute the reaction mixture with 180 μl of TE (cDNA pool; see Hint #4). B. Amplification of the Target cDNA 1. Use 1 to 10 μl aliquots from the above cDNA pool and add the following:     3. Combine 10 μl aliquots of the PCR products with 5 μl aliquots of Sucrose Loading Dye. 4. Load the samples on an Agarose Gel for analysis of the reaction products (see Hint #6, and see Protocol ID#1265).

Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

DEPC-ddH2O DEPC-ddH2O DEPC-ddH2O    Shake or stir the solution Autoclave for 15 min to inactivate the DEPC (CAUTION! See Hint #1) Also see Hint #2 Allow solution to sit for at least 12 hr DEPC-ddH2O    0.1% (v/v) Diethyl Pyrocarbonate (DEPC) in ddH2O Adaptor    50 ng/μl Adaptor 5'-GAC TCG AGT CGA CAT CG-3' 10 mM dNTPs    10 mM dATP 10 mM dTTP 10 mM dGTP 10 mM dCTP Gene-specific Primer    50 ng/μl anti-sense primer to gene of interest 10X PCR Buffer    15 mM MgCl2 500 mM KCl 100 mM Tris-Cl, pH 8.3 DEPC-ddH2O (dT)17-Adaptor    5'-GAC TCG AGT CGA CAT CGA TTT TTT TTT TTT TTT TT-3' 100 ng/μl (dT)17-Adaptor Sucrose Loading Dye (6X)    0.25% (w/v) Xylene Cyanol 0.25% (w/v) Bromophenol Blue 40% (w/v) Sucrose 2 mM dNTPs    2 mM dGTP 2 mM dCTP 2 mM dATP 2 mM dTTP 10 mM DTT

BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

AMV Reverse Transcriptase dCTP RNasin dTTP dGTP Sucrose dATP Magnesium Chloride Isoamyl Alcohol Chloroform Phenol Potassium Cacodylate Tris Potassium Chloride Diethyl Pyrocarbonate (DEPC) DTT Cobalt Chloride (dT)17-Adaptor DNA Polymerase, Taq Bromophenol Blue Oligonucleotide Xylene Cyanol

Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions. 2. To minimize degradation of RNA by RNases, wear gloves when handling samples and reagents, and change gloves regularly while working. Treat water and solutions with DEPC (diethyl pyrocarbonate) to inactivate RNases, and use solutions prepared with RNase-free water and equipment. For more information on working with RNA, see BioTools under the heading of Working With RNA. 3. AMV Reverse Transcriptase is used because it is more stable at higher temperatures than other reverse transcriptases. This allows for the reaction to proceed at 55°C, if so desired, to eliminate potentially inhibitory RNA secondary structures. 4. There is no need to purify the cDNA because the RNase H activity of the AMV Reverse Transcriptase degrades the RNA template. 5. Cycle conditions will depend on your primer, the size of the PCR product, etc. Optimize the PCR for your particular application (see Protocol ID#1242). 6. Southern Blot analysis can also be performed using an internal gene-specific probe.

Citation and/or Web Resources Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

1. Frohman, MA, Dush, MK, Martin, GR. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 1988; 85:8998-9002.