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

5. 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.

7. 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

8. • 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.

9. 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.

10. THANK YOU