Rapid amplification of cDNA ends

As a RACE- PCR ( rapid amplification of cDNA English -ends with polymerase chain reaction), a technique for rapid multiplication of cDNA ends is referred to using the polymerase chain reaction in molecular biology.

The goal of RACE- PCR is the isolation of gene ends and is a modification of the RT-PCR. The gene ends as well as the coding regions translated in transcription into RNA and form the untranslated regions (UTR ) of the transcript. Whereas the start and end of the coding sequence of genes is easily determined with the help of the DNA sequence, the genes ligands are not easy to define. However, the untranslated regions contain essential regulatory elements which affect the translation of the mRNA, and its stability, and are therefore of great interest.

Methods

Using the RACE described here allows the sequence of the untranslated regions of a cytoplasmic mRNA determined. Starting from a known short sequence in the coding region of the transcript can be examined the 5 'end (5'- RACE- PCR), or 3'-end (3'- RACE -PCR) of mRNA. Cytoplasmic mRNA molecules possess a poly (A ) tail, which can be used as a starting point for the 3'- RACE. For 5'-RACE, an adapter to be ligated to the 5 'end of the mRNA. Requirement of both methods is a cDNA synthesis and amplification of cDNA fragments by PCR. Based on the average length of the 3'UTR of 770 nucleotides ( nt), this method is more demanding than the 5 'RACE, in which the 5'UTR is examined by only about 300 nt (Human: 5' 150 nt, 3 ' > 500 nt).

3'RACE

As a starting point for cDNA synthesis for the subsequent 3 'RACE (read: three prime race ) is the poly (A ) tail of eukaryotic cytoplasmic mRNA. In these, an oligonucleotide (primer ) is deposited which consists of an oligo (dT) - strand and a known anchor sequence. The oligo ( dT) primer anneals to the poly (A ) tail and is used as a starting point for cDNA synthesis - the anchor " is 5 ' on'. The RNA is then degraded by RNase H. The cDNA obtained will be used as template in a PCR. For this one uses a primer which binds to the coding region of the gene under investigation in the (known), and the armature, which has been added during the cDNA synthesis. The amplicon can then be further analyzed, for example, sequenced are.

5'RACE

In the 5'-RACE cDNA synthesis starts with a primer that binds in the antisense direction ( antisense ) in the coding region of the mRNA under investigation. Thus, the cDNA comprises a formed portion of the coding sequence and the complete 5 'UTR. Then is added with a terminal and dATP nucleotidyltransferase own poly (A ) end of the cDNA molecule. Subsequently, the mRNA is degraded using RNase H. The product serves as a template to PCR reaction. Here, the genin internal primer and two sense primers are used. The first sense primer consists of an oligo (dT) - and the anchor sequence, the second one only of the anchor sequence. With the aid of the first primer, the anchor is attached to the PCR products, the second provides a specific amplification during PCR. The amplicon can then be further examined, as in the 3'- RACE, eg sequenced are.

RLM -RACE

The so-called CapFinder strategy RACE ( RLM -RACE, RNA ligase mediated RACE) is used to examine the complete 5'-end of the cDNA. For this purpose, first RNA fragments, the free 5 ' phosphate ends wear dephosphorylated by alkaline phosphatase ( calf intestine phosphatase, CIP). Are not affected only by capping protected mRNAs. In the second step, the caps using a nicotinic acid pyrophosphatase ( tobacco acid pyrophoshatase, TAP) are cleaved, the 5' - α - phosphate is retained. Using T4 RNA ligase, an RNA adapter oligonucleotide is then ligated to a known sequence to the mRNA. Can then be carried out using an antisense primer, a cDNA synthesis. This is followed by a PCR with the primers used for cDNA synthesis and appended at the 5 ' end of adapter primer.

SMART RACE

In the SMART RACE (Switching Mechanism at 5 ' end of RNA Transcript ) the total mRNA from an oligo ( dT) primer from transcribed into cDNA. To the 3 ' end of the cDNA (corresponding to the 5' end of the mRNA ), an oligo (dC) is added. With the aid of an anchor primer to a suitable oligo ( dG) and an oligo (dA) - anchor primer of the 3'-end, a PCR is then carried out. Further investigation is analogous to the other RACE methods.