RACE-PCR

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As RACE or RACE-PCR ( English r apid a mplification of c DNA e nds and p Polymerase c hain r eAction ) in which is molecular biology, a combination of methods for the rapid amplification of cDNA of the tails using polymerase chain reaction referred to.

A RACE-PCR is mostly a form or a modification of the RT-PCR with which the ends of genes can be detected. One goal of RACE is often to record a gene as a locus in the genome (as a section in the genetic material). While the start and end points of the coding sequence of genes can usually be determined with the aid of the DNA sequence, the untranslated regions (UTRs) at the two ends of a gene cannot be derived directly from the genome sequence. However, the untranslated regions contain important regulatory elements that influence the translation of the mRNA and its stability and are therefore of great interest.

Methods

The RACE described here can be used to determine the sequence of the untranslated regions of a cytoplasmic mRNA . Starting from a short known sequence in the coding region of the transcript, the 5 'end (5'-RACE-PCR) or 3' end (3'-RACE-PCR) of the mRNA can be examined. Cytoplasmic mRNA molecules have a poly (A) tail , which can be used as a starting point for the 3'-RACE. For the 5'-RACE, an adapter must be ligated to the 5 'end of the mRNA. Both methods require cDNA synthesis and amplification of the cDNA fragments using PCR . Due to 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 of only about 300 nt is examined (humans: 5 'about 150 nt , 3 '> 500 nt).

3'-RACE

The poly (A) tail of a eukaryotic cytoplasmic mRNA serves as the starting point for the cDNA synthesis for the subsequent 3'-RACE (pronounced: 3 prime race). An oligonucleotide ( primer ) consisting of an oligo (dT) strand and a known anchor sequence is attached to this . The oligo (dT) primer attaches to the poly (A) tail and is used as a starting point for cDNA synthesis - the anchor "protrudes 5 'above". The RNA is then degraded by RNase H. The cDNA obtained is used as a template in a PCR . For this purpose, a primer is used that binds in the (known) coding region of the gene under investigation and the anchor that was added during the cDNA synthesis. The amplificate can then be examined further, e.g. B. sequenced .

5'-RACE

In the 5'-RACE , the cDNA synthesis starts with a primer that binds in the antisense direction in the coding area (usually in the ORF ) of the mRNA to be examined. The cDNA formed thus contains part of the coding sequence and the complete 5'UTR. A terminal nucleotidyl transferase and dATP are then used to add a separate poly (A) end to the cDNA molecule. The mRNA is then degraded with the help of RNase H. The product serves as a template for a PCR reaction. The gene-internal primer and two sense primers are used here. The first sense primer consists of an oligo (dT) and anchor sequence, the second only consists of the anchor sequence. The anchor is attached to the PCR products with the help of the first primer, the second ensures a specific amplification during the PCR. As in the 3'-RACE, the amplificate can then be examined further, e.g. B. sequenced .

RLM-RACE

In the RLM-RACE ( R NA l Igase m ediated RACE ) single strands are joined together by a corresponding nucleic acid ligase. The method is used to examine gene ends. Two variants were presented in the original work; one for the 3 'end of the respective gene and one for the 5' end. The following shows the variant for the 5 'end:

  • The 5 'cap structure of the mRNA is chemically removed using sodium periodate (the method is based on). The mRNA then contains more than one phosphate group at the 5 'ends and is therefore dephosphorylated by alkaline phosphatase ( calf intestine phosphatase , CIP). Since exactly one phosphate group is required at the respective 5 'end of the mRNA for the ligation, a kinase is used ( T4 polynucleotide kinase ). With the help of the T4 RNA ligase , short RNA oligonucleotides with a uniform sequence are attached to the 5 'ends of the mRNA molecules. (In the original work, the short RNA oligonucleotides with a uniform sequence came from a previously carried out polymerase reaction in which the T7 RNA polymease was used.) The modified mRNA molecules are now used to create a cDNA Synthesis performed by linking short DNA oligonucleotides of random sequence to the mRNA by complementary base pairing. A reverse transcriptase uses these DNA oligonucleotides (hexamers) as primers , while it uses the respective mRNA molecule as a template for the cDNA first strand synthesis. The cDNA single strands each have a uniform sequence at their 3 'ends. (This uniform sequence goes back to the RNA ligation, which was previously carried out with the short RNA oligonucleotides of uniform sequence.) A cDNA second strand synthesis is not required; the PCR can now be carried out with a uniform and a gene-specific primer.

Oligo capping

The oligo-capping introduced by Maruyama Kazuo and Sugano Sumio in 1994 is similar to the RLM-RACE, which was described by Xiuwen Liu and Martin A. Gorovsky in 1993. In both cases, uniform, artificially generated RNA oligonucleotides are ligated to the 5 'ends of the RNA molecules that represent the biological test material, which are intended to replace the 5' cap structure . Differences exist, among other things, in the type of removal of the cap structure and in the number and order of the steps.

The oligo-capping is used to examine the complete 5 'end of the cDNA. For this purpose, RNA fragments that have free 5 'phosphate ends are first dephosphorylated by alkaline phosphatase ( bacterial alkaline phosphatase , BAP). Only mRNAs protected by capping are not affected by this. In the second step, the caps are split off with the help of nicotinic acid pyrophosphatase ( tobacco acid pyrophosphatase , TAP), whereby the 5'-α-phosphate is retained. Using the T4 RNA ligase, an RNA adapter oligonucleotide with a known sequence is then ligated to the mRNA. A cDNA synthesis can then be carried out with an antisense primer. This is followed by a PCR with the primer used for cDNA synthesis and the adapter primer attached to the 5 'end.

SMART RACE

The term SMART ™ ( s witching m echanism a t 5 'end of R NA t ranscript ) is a first by Clontech Laboratories (Inc.) unused trade name . The forerunner of SMART ™ was CapFinder ™. Today SMART products, e.g. B. the "SMART® cDNA Library Construction Kit" sold by Takara Bio (Inc.).

In SMART-RACE, the entire mRNA is transcribed into cDNA starting from an oligo (dT) primer. The reverse transcriptase attaches a short oligo (dC) sequence to the 3 'end of the cDNA opposite the CAP structure (corresponds to the 5' end of the mRNA), which is initially not paired in a complementary manner. With the help of another anchor primer, which has a suitable oligo (dG) structure for a complementary pairing, the reverse transcriptase can lengthen the cDNA strand by switching from the mRNA as a template to the anchor primer. The cDNA single strand that is now available has specific sequences at both ends due to the anchor primers used, which are suitable for a RACE-PCR, which is then carried out. The further investigation is analogous to the other RACE methods.

See also

literature

  • MA Frohman, MK Dush, GR Martin: Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. In: Proceedings of the National Academy of Sciences . tape 85 , no. 23 , December 1, 1988, ISSN  0027-8424 , p. 8998-9002 , doi : 10.1073 / pnas.85.23.8998 , PMID 2461560 , PMC 282649 (free full text).
  • MA Frohman: Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE. In: Methods Enzymol. Volume 218, 1993, pp. 340-356, PMID 7685466 .

Individual evidence

  1. ↑ Pickering, BM & Willis, AE (2004): The implications of structured 5 'untranslated regions on translation and disease. In: Semin Cell Dev Biol. Vol. 16, pp. 39-47. PMID 15659338 .
  2. ↑ a b Mazumder, B. et al. (2003): Translational control by the 3'-UTR: the ends specify the means. In: Trends Biochem. Sci. Vol. 28, pp. 91-98. PMID 12575997 doi: 10.1016 / S0968-0004 (03) 00002-1 .
  3. ↑ Scotto-Lavino E., Du G. & Frohman MA (2007): 3 'End cDNA amplification using classic RACE In: Nature Protocols. Vol. 1, pp. 2742-2745 doi: 10.1038 / nprot.2006.481 .
  4. ↑ Nature Methods 2, 629-630 (2005) doi: 10.1038 / nmeth0805-629
  5. ↑ a b c d e Xiuwen Liu, Martin A. Gorovsky: Mapping the 5 ′ and 3 ′ ends of Tetrahymena thermophila mRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE) . In: Nucleic Acids Research . tape 21 , no. 21 , 1993, ISSN  0305-1048 , pp. 4954-4960 , doi : 10.1093 / nar / 21.21.4954 , PMID 8177745 , PMC 311412 (free full text).
  6. ↑ Jay R. Greenberg, Virginia E. Burn: Proteins associated with rabbit reticulocyte mRNA caps during translation as investigated by photocrosslinking . In: Nucleic Acids Research . tape 16 , no. 8 , 1988, ISSN  0305-1048 , pp. 3437-3454 , doi : 10.1093 / nar / 16.8.3437 , PMID 3131737 , PMC 336504 (free full text).
  7. ↑ Maruyama Kazuo, Sugano Sumio: Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides . In: Genes . tape 138 , no. 1-2 , January 1994, pp. 171-174 , doi : 10.1016 / 0378-1119 (94) 90802-8 .
  8. ↑ Clontech Laboratories: SMART ™ cDNA Amplification Construction Kit . In: CLONTECHniques . tape 16 , no. 4 , October 2001, p. 12–13 (PDF file online - web location: http://www.ebiotrade.com/emgzf/clnt/ctqoct01.pdf ; accessed June 13, 2020).
  9. ↑ Clontech Laboratories: SMART ™ cDNA Amplification Construction Kit . In: CLONTECHniques . tape 14 , no. 3 , 1999, p. 4-7 .
  10. ↑ Clontech Laboratories: CapFinder ™ PCR cDNA Library Construction Kit . In: CLONTECH (ed.): CLONTECHniques . tape 11 , no. 1 , 1996, p. 2-4 .
  11. ↑ Takara Bio USA: SMART® cDNA Library Construction Kit User Manual. Accessed June 13, 2020 (PDF file online).