Demystifying RACE

Oct 16 '07
Rapid Amplification of cDNA Ends, or RACE, is the amplification of cDNA sequences from mRNA using a defined internal site and an unknown 3’ or 5’end. First, mRNA is reverse transcribed (RT) to cDNA. The 3’ kits use a simpler protocol taking advantage of the natural poly(A) tail as a priming site. The 3’ region is amplified using a kit supplied adapter primer that anneals to the poly(A) tail region. To amplify the 5’ end the most common procedure involves adding homopolymeric tails to the 3’ end of the cDNA using terminal deoxynucleotide transferase (TdT). This tail creates a binding site for the kit supplied anchor primer. PCR amplification using the gene specific nested primer and the anchor primer yields a fragment containing the 5’ end of the gene. Another method uses a kit primer during RT that produces cDNA with a phosphorylated 5’ end. The resulting single stranded cDNA is circularized such that the unknown region can be amplified using gene specific primers within the known region. A larger unknown region can be mapped using this protocol. One procedure takes advantage of the eukaryotic mRNA cap. The 5’phosphate group is removed from uncapped partial mRNA. The cap is then removed from complete transcripts such that the kit supplied RNA oligo can ligate to the exposed 5’phosphate. After RT, the unknown region can be amplified using the primer specific to the RNA oligo. A few things need to be determined before deciding which kit is right for your situation. First, is it the 5’ or 3’ end that needs to be defined? If it is eukaryotic DNA, the 5’ cap can be easily utilized. If the sequence is known, a primer can be designed within 100 bp to the presumed 5’ end, to use the homopolymeric tail protocol. If little is known of the sequence, adding the 5’ phosphate to the mRNA may be the most efficient answer.

Katrina S.Stumpf, MS
Research Scientist
McGuire VA Medical Center

Company	Invitrogen	Invitrogen	Invitrogen	Roche Applied Science	Takara Bio USA
Item	3' RACE System for Rapid Amplification of cDNA Ends	5' RACE System for Rapid Amplification of cDNA Ends Version 2.0	GeneRacer™ Kit with SuperScript™ II RT and TOPO TA Cloning kit for Sequencing	RACE Kit, 2nd Generation; New formulation containing recombinant Terminal Transferase and recombinant AMV-RT	RACE Core Set, 5´-Full
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Description	3' RACE System is suitable for rapid amplification of cDNA ends (RACE) (1-3) and anchored PCR between a defined point within mRNA and the 3' poly(A) end (Figure 1). The system is useful for the amplification of rare messages for which little sequence information is available and for capturing the 3' end information of... More...	The 5' RACE System Version 2.0 is suitable for rapid amplification of cDNA ends (RACE) (1) and anchored PCR (2) between a defined point in the mRNA and the 5' end. The system is useful for the amplification of rare messages for which little sequence information may be available and for capturing the 5' end information of mRNA... More...	GeneRacer™ is an advanced RACE (rapid amplification of cDNA ends) technique that improves the efficiency of amplifying full-length 5' and 3' cDNA ends. With the GeneRacer™ Kit you can:	Obtaining a full-length cDNA is of critical importance for structural and expression studies. The amplification of DNA sequences from a messenger RNA template between a defined internal site and unknown sequences of either the 3' or the 5'end of the mRNA are often referred as RACE (rapid amplification of cDNA ends) anchored PCR or one-sided... More...	Reverse transcription of mRNA followed by PCR is a common method for obtaining cDNA clones for expression analysis. However, it can be difficult to obtain full-length cDNA by this method. Rapid amplification of cDNA ends (RACE) helps overcome this problem by using a gene-specific primer for reverse transcription, followed by amplification with specially designed... More...
Catalog Number	18373019	18374058	L150201	3353621001	TAK 6122
Quantity	20 rxns	10 rxns	1 kit	1 kit (10 reactions)	10 rxn
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