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 3f or 5f 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
3f
RACE takes advantage of the natural poly(A) tail found in mRNA as a 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 3f-mRNA sequences
that lie between the exon and the poly(A) tail. In this section, two protocols,
one with the GeneRacer kit (Invitrogen) 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 5f and 3f ends of cDNA
using known cDNA sequence.@ The kit
ensures the amplification of only full-length transcripts via elimination of
truncated 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 5f ends of decapped 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 3f to GSP1 and
to contain a CAUCAUCAUCAU sequence at its 5f 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 cells
E LB plates
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 probably not so highly-purified.@ If the RNA solution contains insoluble material, use the supernatant obtained after brief centrifugation.
1)-2
Incubate at 70˚C for 10 min, and then place on 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 hr 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
Place the 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 check
yield and specificity of your RACE product, perform nested PCR with GSP2 and
UAP primers.
3)
UDG cloning using pIMAz2 and sequencing
3)-1
Separate PCR product by electrophoresis with 1 % (w/v) agarose (SeaKem GTG,
TaKaRa) and recover a target product with a QIAquick Gel Extraction kit
(QIAGEN) or equivalent.
3)-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
@@@ make up to 20
µl with nuclease-free water.
3)-3
Incubate at 37˚C for 30 min. Place on ice.
3)-4
After annealing, 2-4 µl of the annealing reaction should be used for
transformation.
3)-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
5f
RACE is a technique that facilitates isolation and characterization of 5f ends
from low copy messages. In this section, a procedure based on the 5f 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 3f 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 3f 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 5f 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 3f to GSP1 and
to contain a CAUCAUCAUCAU sequence at its 5f 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 cells (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 probably not so highly-purified.@ If RNA solution contains insoluble material,
use the supernatant obtained 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, not been available more recently.@ Probably, SizeSep 400 Spun Columns (Amersham Bioscience) and QiaQuick PCR purification kit (Qiagen) can be used instead.
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 ensure 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.@ Place
on 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 5f
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.