How can we achieve cloning of mRNA's complete sequence?

In RACE technique, PCR is used to amplify partial region between a single point in a mRNA transcript and its 3'- or 5'-end. The premise is that a short internal stretch of the sequence from the target mRNA is already known. Extension of the partial cDNAs from the unknown end of the message back to the known region is achieved by 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) should be carried out by 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.

According to statistics in the past, most attempts to identify and isolate a novel cDNA will result in clones that represent only a part of the mRNA's complete sequence. Then how could we achieve cloning of mRNA's complete sequence?

The answer should be PCR in RACE technique. The missing sequence (cDNA ends) can be cloned by PCR, using the technique called rapid amplification of cDNA ends (RACE).

RACE cloning has several advantages for mRNA target cloning:

 It takes only weeks to screen cDNA libraries,
 Can obtain individual cDNA clones
 Can analyze the clones to determine if the missing sequence is present and such information can be generated within a few days.                          

Because of those advantages, RACE cloning has becomes popular in the application to modify RNA preparation and/or reverse transcription conditions until full-length cDNAs are generated and observed.

The principle of RACE technique


Creative Biogene’s RACE kits

Creative Biogene’s RACE kits are specially designed for 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. The 5' RACE kits contain all necessary components for performing 5' RACE. The kits can achieve the amplification of rare messages with little sequence information. And the PCR products can be used for sequencing and cloning.

More information at http://www.creative-biogene.com/5-RACE-RTRZ-02-1232898-71.html