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RACE (biology)

 

 
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RACE (biology)
 
 
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RACE, or Rapid Amplification of cDNA Ends, is a technique used in molecular biology
Molecular biology

Molecular biology is the study of biology at a molecule level. The field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry....
 to obtain the full length sequence of an RNA
RNA

Ribonucleic acid is a type of molecule that consists of a long chain of nucleotide units. Each nucleotide consists of a nucleobase, a ribose sugar, and a phosphate....
 transcript found within a cell. RACE results in the production of a cDNA copy of the RNA sequence of interest, produced through reverse transcription
Reverse transcription

Reverse transcription is the process of making a double stranded DNA molecule from a single stranded RNA template. It is called reverse transcription as it acts in the opposite or reverse direction to transcription ....
, followed by PCR amplification of the cDNA copies. The amplified cDNA copies are then sequenced to obtain a full length sequence of the original RNA.






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RACE, or Rapid Amplification of cDNA Ends, is a technique used in molecular biology
Molecular biology

Molecular biology is the study of biology at a molecule level. The field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry....
 to obtain the full length sequence of an RNA
RNA

Ribonucleic acid is a type of molecule that consists of a long chain of nucleotide units. Each nucleotide consists of a nucleobase, a ribose sugar, and a phosphate....
 transcript found within a cell. RACE results in the production of a cDNA copy of the RNA sequence of interest, produced through reverse transcription
Reverse transcription

Reverse transcription is the process of making a double stranded DNA molecule from a single stranded RNA template. It is called reverse transcription as it acts in the opposite or reverse direction to transcription ....
, followed by PCR amplification of the cDNA copies. The amplified cDNA copies are then sequenced to obtain a full length sequence of the original RNA. 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.

The first step in RACE is to use a reverse transcription-polymerase chain reaction
Polymerase chain reaction

The polymerase chain reaction is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzyme DNA replication....
 (RT-PCR) to produce a cDNA copy of a region of the RNA transcript. In this process, an unknown end portion of a transcript is copied using a known sequence from the center of the transcript. The copied region is bounded by the known sequence, and either the 5' or 3' end.

The protocols for 5' or 3' RACE differ slightly. 5' RACE-PCR begins using mRNA as a template for a first round of cDNA synthesis (or reverse transcription
Reverse transcription

Reverse transcription is the process of making a double stranded DNA molecule from a single stranded RNA template. It is called reverse transcription as it acts in the opposite or reverse direction to transcription ....
) reaction using an anti-sense (reverse) oligonucleotide
Oligonucleotide

An oligonucleotide is a short nucleic acid polymer, typically with twenty or fewer nucleotide. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized by polymerizing individual nucleotide precursors....
 primer that recognizes a known sequence in the gene of interest; the primer
Primer (molecular biology)

A primer is a strand of nucleic acid that serves as a starting point for DNA replication. They are required because the enzymes that catalyze replication, DNA polymerases, can only add new nucleotides to an existing strand of DNA....
 is called a gene specific primer (GSP), and it copies the mRNA template in the 3' to the 5' direction to generate a specific single-stranded cDNA product. Following cDNA synthesis, the enzyme
Enzyme

Enzymes are biomolecules that catalysis chemical reactions. Almost all enzymes are proteins. In enzymatic reactions, the molecules at the beginning of the process are called Substrate , and the enzyme converts them into different molecules, the products....
 terminal deoxynucleotidyl transferase
Terminal Deoxynucleotidyl Transferase

Terminal Deoxynucleotidyl Transferase, also known as TdT and terminal transferase, is a specialized DNA polymerase expressed in immature, pre-B, pre-T lymphoid cells, and acute lymphoblastic leukemia/lymphoma cells....
 (TdT) is used to add a string of identical nucleotide
Nucleotide

Nucleotides are molecules that comprise the structural units of RNA and DNA. Additionally, nucleotides play central roles in metabolism. In that capacity, they serve as sources of chemical energy , participate in cell signaling , and are incorporated into important cofactors of enzymatic reactions ....
s, known as a homopolymeric tail, to the 3' end of the cDNA. A PCR
Polymerase chain reaction

The polymerase chain reaction is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzyme DNA replication....
 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.

3' RACE-PCR uses the natural polyA tail that exists at the 3' end of all eukaryotic mRNAs for priming during reverse transcription, so this method does not require the addition of nucleotides by TdT. cDNAs are generated using an Oligo-dT-adaptor primer that complements the polyA
Polyadenylation

Polyadenylation is the addition of a poly tail, a stretch of RNA where all the nucleobase are adenines, onto an RNA molecule. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA for translation ....
 stretch and adds a special adaptor sequence to the 3' 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.