4.5@ RACE

Yuji Hiwatashi, Keiko Sakakibara, Rumiko Kofuji, and Tomoaki Nishiyama

 

Introduction

RACE (Rapid Amplification of cDNA Ends) is a procedure for amplification of nucleic acid sequence from a mRNA template between a defined internal site and unknown sequences at either the 3f and 5f end of the mRNA.@ In general, PCR amplification of relatively few target molecules in a complex mixture requires two sequence-specific primers that flank the region of sequence to be amplified.@

 

3'RACE

3f RACE takes advantage of the natural poly(A) tail found in mRNA as generic priming site for PCR amplification. In this procedure, mRNAs are converted into cDNA using reverse transcriptase (RT) and an oligo-dT adapter primer. Specific cDNA is then directly amplified by PCR using a gene-specific primer (GSP) that anneals to a region of known exon sequences and an adapter primer that targets the poly(A) tail region. This permits the capture of unknown 3f-mRNA sequences that lie between the exon and the poly(A) tail. In this section, two protocols, one with GeneRacer kit (Invtrogen) and another with 3'RACE System for Rapid Amplification of cDNA Ends (Invitrogen), are described.

 

1. GeneRacer kit (Invitrogen)

The GeneRacer kit provides a method to obtain full-length 5f and 3f ends of cDNA using known cDNA sequence.@ The kit ensures the amplification of only full-length transcripts via elimination of truncate messages formed during the amplification process. This technique is based on RNA ligase-mediated and oligo-capping rapid amplification of cDNA ends (RACE) methods, and results in the selective ligation of an RNA oligonucleotide to the 5f ends of decapping mRNA using T4 RNA ligase.

@

Solution required

E Physcomitrella patens total RNA

E SuperScript III Reverse Transcriptase

E dNTP

E 5x first strand buffer

E RNace out (RNase inhibitor)

E GeneRacer oligo dT primer

E TaKaRa Ex Taq Hot Start version

E PCR buffer

E dNTP

E GeneRacer 3' primer

E GeneRacer 3' nested primer

E Gene-specific primer

 

Procedure

Please refer to the appropriate manual included with this kit.

 

2. 3'RACE System for Rapid Amplification of cDNA Ends (Invitrogen)

Solution required

E Physcomitrella patens total RNA

E SuperScript II Reverse Transcriptase (Invitrogen)

E Adaptor Primer: ggCACgCgTCgACTAgTACT(17) (Invitrogen)

E Anchor primer: CUACUACUACUAGGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG

E Universal Amplification Primer (UAP) : CUACUACAUCAUggCCACgCgTCgACTAgTAC:

E GSP1

E GSP2: nested gene-specific primer. This primer is designed to anneal 3f to GSP1 and to contain a CAUCAUCAUCAU sequence at its 5f end.

E TaKaRa Ex Taq

E UDG (1units/µl)

E UDG cloning vector pIMAz2 (Hiwatashi et al. 2001)
a) Digest pGEM 3z with SmaI and than amplify with UAGUAGUAGUAGcccgggtaccgagctcgaa and AUGAUGAUGAUGcctctagagtcgacctgcaggca using the SmaI-digested pGEM3za as template.@ Then, purify with QIAGEN Qiaquick PCR purification kit or equivalents.@ Use this fragment, named pIMAz2, for UDG cloning.@

E DH5alpha competent cell

E LB plate containing 50 µg/ml ampicillin

E QIA quick Gel extraction kit or Gene Clean II kit (BIO101), etc:@

E Forward Primer: CCCAGTCACGACGTTGTAAACG

E Reverse Primer: AGCGGATAACAATTTCACACAGG

 

Procedure

1) First strand cDNA

1)-1 Add the following reagents to a micro tube and mix by pipetting.@

@@@ 3 µg total RNA*1

@@@ 1 µl Adaptor Primer (100mol/ul)

@@@ fill up to 11 µl with nuclease-free water.

*1@ You can use total RNA extracted with Isogen (Nippon gene), which is likely not so high-purified.@ If RNA solution contains insoluble material, use supernatant after brief centrifugation.

1)-2 Incubate at 70˚C for 10 min, and then remove ice.@

1)-3 Add the following reagents to the microtube and mix by pipetting.
@@@ 4 µl@ 5x 1st strand Buffer
@@@ 2 µl@ 0.1 M DTT
@@@ 1 µl@ 10 mM dNTP

1)-4 Incubate at 37˚C for 2 min

1)-5 Add 1 ul of 200 units/µl SuperScript II Reverse Transcriptase and mix by pipetting.

1)-6 Incubate at 42˚C for 1 hrs to synthesize cDNA.

2) PCR amplification

2)-1 Add the following reagents in a PCR tube.
@@@ 2 µl@ cDNA solution
@@@ 2 µl@ Ex Taq Buffer
@@@ 2 µl@ 2.5 mM dNTP

@@@ 0.5 µl 10 pmol/µl GSP1

@@@ 0.5 µl 10 pmol/µl Anchor
@@@ 0.125 µl@ Ex Taq (5 U/ul)@

@@@ 17.5 µl@ nuclease-free water

2)-2 Set a tube in a PCR thermal cycler.

2)-3 Perform the following cycle.@
@@@@ 94˚C, 5 min x 1 cycle
@@@@ 94˚C, 30 sec- > 52-68˚C*6, 30 sec -> 72˚C, 2 min x 25-35 cycles*7
@@@@ 72˚C, 5 min
@@@@@@ *6 Tm (GSP1) - 5
@@@@@@ *7 Use 25-35 cycles depending on the transcript abundance.@

2)-4 To yield and specificity of your RACE product, perform nested PCR with GSP2 and UAP primers.@

 

5) UDG cloning using pIMAz2 and sequencing

5)-1 Separate PCR product by electrophoresis with 1 % (w/v) agarose (SeaKem GTG, TaKaRa) and recover a target product with QIAquick Gel Extraction kit (QIAGEN) or equivalents.@

5)-2 Add the following to a tube

@@@ 1-5 µl@ 10-50 ng PCR product

@@@ 2 µl@ 25 ng/µl pIMAz2

@@@ 2 µl@ 10x reaction buffer (200 mM Tris-Hcl (pH8.4), 500 mM KCl)

@@@ 1 µl 1 units/µl UDG

fill up to 20 µl with nuclease-free water.

5)-3 Incubate at 37˚C for 30 min.@ Remove to ice.

5)-4 After annealing, 2-4 µl of the annealing reaction should be used for transformation.@@

5)-5 Once you have cloned your PCR products from selected transformants, you will isolate plasmid DNA using your method of choice. You should select 10-12 clones for sequencing. To sequence your PCR products using pIMAz2, please refer to the appropriate manual. You can obtain the sequence information using reverse and forward primers.

 

 

5'RACE

5f RACE is a technique that facilitates isolation and characterization of 5f ends from low copy messages. In this section, a procedure based on 5f RACE system of Invitrogen is described. First strand cDNA synthesis is primed using a gene-specific antisense oligonucleotide (GSP1). This permits cDNA conversion from specific mRNA or related families of mRNAs. Following cDNA synthesis, the first strand product is purified from unincorporated dNTPs and GSP1. TdT is used to add homopolymeric tails to the 3f ends of the cDNA. Tailed cDNA is then amplified by PCR using a mixture of three primers. First, a nested gene-specific primer (GSP2), which anneals 3f to GSP1 and a complementary homopolymer-containing anchor primer are used. Then, the GSP2 primer and a corresponding adapter primer, which anneals to the anchor primer, are used. This allows amplification of unknown sequences between the GSP2 and the 5f end of the mRNA.

 

5'RACE System for Rapid Amplification of cDNA Ends (Invitrogen)

Solution required

E Physcomitrella patens total RNA

E SuperScript II Reverse Transcriptase (Invitrogen)

E 10xReaction Buffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl]

E 25 mM MgCl2

E 10 mM dNTP

E RNase H (Invitrogen)

E Glass Max DNA isolation spin cartridge (Invitrogen)

E Terminal deoxynucleotidyl transferase (TdT) (Invitrogen)

E 2mM dCTP

E Anchor Primer: CUACUACUACUAggCCACgCgTCgACTAgTACgggIIgggIIgggIIg

E Universal Amplification Primer: CUACUACAUCAUggCCACgCgTCgACTAgTAC

E GSP1: gene-specific anti-sense primer

E GSP2: nested gene-specific primer. This primer is designed to anneal 3f to GSP1 and to contain a CAUCAUCAUCAU sequence at its 5f end.

E TaKaRa Ex Taq

E UDG cloning vector pIMAz2 (Hiwatashi et al. 2001)
a) Digest pGEM 3z with SmaI and than amplify with UAGUAGUAGUAGcccgggtaccgagctcgaa and AUGAUGAUGAUGcctctagagtcgacctgcaggca using the SmaI-digested pGEM3za as template.@ Then, purify with QIAGEN Qiaquick PCR purification kit or equivalents.@ Use this fragment, named pIMAz2, for UDG cloning.@

E UDG (1 units/µl: Invitrogen)

E High competent cell (XL10Gold)

E LB plate supplemented with 50 µg/ml ampicillin.

E Qiaquick Gel Extraction kit (QIAGEN) or equivalents

E Forward Primer: CCCAGTCACGAVGTTGTAAACG

E Reverse Primer: AGCGGATAACAATTTCACACAGG

 

Procedure

1)@ First strand cDNA synthesis

1)-1 Add the following reagents to the tube, mix by pipetting, and centrifuge briefly.
1 µl@ total RNA*1
1 µl@ GSP1 (2.5 mol/µl)
fill up to 15 ul with nuclease-free water.
*1@ You can use total RNA extracted with Isogen (Nippon gene), which is likely not so high-purified.@ If RNA solution contains insoluble material, use supernatant after brief centrifugation.

1)-2 Heat at 70˚C for 10 min, then place on ice for 3 min.@

1)-3 Add the following reagents in the tube and mix by pipetting.
@@2.5 µl@ Reaction Buffer
@@3 µl@ 25 mM MgCl2
@@2.5 µl@@ 0.1 M DTT
@@1 µl 10 mM dNTP

1)-4 Incubate at 42˚C for 2 min.

1)-5 Add 1 µL of 200 u/ µl SuperScript II Reverse Transcriptase, and mix by pipetting.@

1)-6 Incubate at 42˚C for 1 hrs to synthesize first strand cDNA.@

1)-7 Incubate at 70˚C for 15 min to denature reverse Transcriptase.

1)-8 Incubate 55˚C for 2 min.@

1)-9 Add 1 µl of RNase H and mix by pipeting.

1)-10 Incubate at 55˚C for 10 min and then place at 4˚C.@@

2) Purification of first strand cDNA

Prior to TdT tailing, excess nucleotides and GSP1 must be removed from the first strand product.@ For purification of the first strand cDNA, we had used GLASSMAX DNA isolation Spin Cartridge (GIBCO).@ This kit has, however, been not available.@ Probably, SizeSep 400 Spun Columns (Amersham Bioscience) and QiaQuick PCR purification kit (Qiagen) can be used.@@

 

3) Homopolymeric tailing of cDNA

3)-1 Add the following reagents in a tube, and mix gently.@
@@@ 10 µl cDNA solution
@@@ 2.5 µl 10xReaction Buffer
@@@ 1.5 µl 25 mM MgCl2
@@@ 2.5 µl 2 mM dCTP

@@@ 7.5 µl nuclease-free water

3)-2 Heat at 94˚C for 3 min, then place on ice.

3)-3 Add 1 µl of TdT, and mix gently

3)-4 Incubate at 37 ˚C for 10 min.@

3)-5 Incubate at 65˚C for 10 min, and then at 4˚C

 

4) PCR amplification

4)-1 Add the following reagents in a PCR tube.
@@@ 2.5 µl@ Tailed cDNA solution
@@@ 2.5 µl@ 10x Ex Taq Buffer
@@@ 0.5 µl@ 10 mM dNTP
@@@ 0.125 µl@ Ex Taq (5 U/µl)@

@@@ 17.5 µl@ nuclease-free water

4)-2 Set a tube in a PCR thermal cycler.@ Heat at 95˚C for 1 min, then at 80˚C for 1 min.

4)-3 Add 1 µl of Anchor Primer (10 pmol/µl) and 1 µl of GSP2 (10 pmol/µl) in the tube.

4)-4 Perform the following cycle.@
@@@@ 94˚C, 5 min x 1 cycle
@@@@ 94˚C, 30 sec- > 52-68˚C (*6), 30 sec -> 72˚C, 2 min x 25-35 cycles (*7)
@@@@ 72˚C, 5 min
@@@@@@ *6 Tm (GSP2) - 5
@@@@@@ *7 Use 25-35 cycles depending on the transcript abundance.@ To yield and specificity of your RACE product, perform nested PCR.@

 

5) UDG cloning using pIMAz2 and sequencing

5)-1 Separate PCR product by electrophoresis with 1 % (w/v) agarose (SeaKem GTG, TaKaRa) and recover a target product with QIAquick Gel Extraction kit (QIAGEN) or equivalents.@

5)-2 Add the following to a tube

@@@ 1-5 µl@ 10-50 ng PCR product

@@@ 2 µl@ 25 ng/µl pIMAz2

@@@ 2 µl@ 10x reaction buffer (200 mM Tris-Hcl (pH8.4), 500 mM KCl)

@@@ 1 µl 1 units/µl UDG

fill up to 20 µl with nuclease-free water.

5)-3 Incubate at 37˚C for 30 min.@ Remove to ice.

5)-4 After annealing, 2-4 µl of the annealing reaction should be used for transformation.@@

5)-5 You should select 10-12 clones for sequencing to ensure full coverage of the 5f end. Many genes have alternative start sites for transcription and splice variants. To sequence the PCR products using pIMAz2, refer to the appropriate manual. You can obtain the sequence information using reverse and forward primers.