Rapid amplification of c-DNA ends

Information about Rapid amplification of c-DNA ends

Published on June 8, 2016

Author: lovnishthakur75

Source: slideshare.net


1. Rapid amplification of c-DNA ends By-: Lovnish Thakur ASU2014010100099 INTEGRATED BIOTECH(4TH SEM)

2. Rapid amplification of cDNA ends is a technique used in molecular biology to obtain the full length sequence of an RNA transcript found within a cell RACE results in the production of a cDNA copy of the RNA sequence of interest, produced through reverse transcription, followed by PCR amplification of the cDNA copies The amplified cDNA copies are then sequenced and, if long enough, should map to a unique genomic region. RACE can provide the sequence of an RNA transcript from a small known sequence within the transcript to the 5' end (5' RACE-PCR) or 3' end (3' RACE-PCR) of the RNA. This technique is sometimes called one-sided PCR or anchored PCR

3. Step 1. use reverse transcription to produce a cDNA copy of a region of the RNA transcript 2. In this process, an unknown end portion of a transcript is copied using a known sequence from the center of the transcript 3. The copied region is bounded by the known sequence, at either the 5' or 3' end The only requirement for RACE cDNA amplification is that you know at least 23– 28 nucleotides of sequence information in order to design gene-specific primers (GSPs) for the 5'- and 3'-RACE reactions

4. 3' RACE-PCR • 3' RACE-PCR uses the natural polyA tail that exists at the 3' end of all eukaryotic mRNAs for priming during reverse transcription • does not require the addition of nucleotides by TdT. • cDNAs are generated using an Oligo-dT-adaptor primer (a primer with a short sequence of deoxy-thymine nucleotides) that complements the polyA stretch • And adds a special adaptor sequence to the 5' end of each cDNA. • PCR is then used to amplify 3' cDNA from a known region using a sense GSP, and an anti-sense primer complementary to the adaptor sequence.

5. 5' RACE-PCR • begins using mRNA as a template for a first round of cDNA reaction using an oligonucleotide primer that recognizes a known sequence in the middle of the gene of interest; the primer is called a gene specific primer (GSP) • The primer binds to the mRNA, and the enzyme reverse transcriptase adds base pairs to the 3' end of the primer to generate a specific single- stranded cDNA

6. • Following cDNA synthesis, the enzyme terminal deoxynucleotidyl transferase (TdT) is used to add a string of identical nucleotides, known as a homopolymeric tail, to the 3' end of the cDNA. • A PCR reaction is then carried out, which uses a second anti-sense gene specific primer (GSP2) that binds to the known sequence, and a sense (forward) universal primer (UP) that binds the homopolymeric tail added to the 3' ends of the cDNAs to amplify a cDNA product from the 5' end.

7. Reference • "Rapid amplification of 5' cDNA ends," in Molecular Cloning: A Laboratory Manual (eds. Sambrook, J. & Russell, D.W.) Chapter 8 Protocol 9, 8.54−8.60 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2001) • Principles of Gene Manipulation and Genomics, S. B. Primrose and R. M. Twyman, Blackwell Publishing, 2006 (7th ed.), pages 111-12.


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