4.3@ Isolation of genomic fragment by TAIL-PCR

Tomomichi Fujita, Yuji Hiwatashi and Tomoaki Nishiyama

Literatures for TAILiThermal asymmetric interlacedj-PCR

Liu, Y.G. and Whttier, R.F. (1995) Thermal asymmetric interlaced PCR: Automatable amplification and sequncing of insert and fragments from P1 and YAC clones for chromosome walking. Genomics 25, 74-681.

Liu, Y.G., Mitsukawa, N., Oosumi, T. and Whttier, R.F. (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J. 8, 457-463.

We describe (1) TAIL-PCR with Ex Taq DNA polymeraseiTAKARA, Kyotoj, and (2) TAIL-PCR with KOD Dash DNA polymerase (TOYOBO, Osaka) to shorten the time it takes.@ To get a longer fragment, Ex Taq is superior to KOD Dash.@ We use GeneAmp 9600iPerkin-Elmerj thermal cycler.@ Different PCR cycler may require optimization of the PCR conditions.

 

arbitrary primer

gene specific primer

Design GSP with Tm greater than 72C.

More than 50bp must be remained downstream of a third gene specific primer to check if a gained TAIL PCR fragment is correctly extended from an original sequence.

(1) TAIL-PCR with Ex Taq DNA Polymerase

1st PCR reaction mix (total 20µl)

mix solution on ice

use 96-well plate if necessary

1.00 µl

40-200 ng/µl genomic DNA

2.00 µl

10x ExTaq buffer

1.60 µl

2.5 mM dNTPs

0.40 µl

10 pmol/µl GSP1

1.00 µl

100 pmol/µl A1-3 (degenerate primer)

0.20 µl

5 U/µl ExTaq

13.80 µl

H2O

1st PCR reaction

Place a PCR plate from ice after a temperature of heat block is more than 80C.

95˚C, 1min
(94˚C, 30 s -> 65˚C, 30 s -> 72˚C, 3 min 30 s) x 5
94˚C, 30 s -> 30˚C, 30 s -> 72˚C, 3 min 30 s
94˚C, 30 s -> 68˚C, 30 s -> 68˚C, 3 min 30 s@ „ª„­
94˚C, 30 s -> 68˚C, 30 s -> 68˚C, 3 min 30 s@@ x 13
94˚C, 30 s -> 44˚C, 30 s -> 68˚C, 3 min 30 s@ „ª„®
68˚C, 5min
4˚C

 

2nd PCR reaction (total 20 µl)

1.00 µl

1st PCR product (DF=50-200)

2.00 µl

10x ExTaq buffer

1.60 µl

2.5mM dNTPs

0.40 µl

10 pmol/µl GSP2

0.80 µl

100 pmol/µl A1-3 (degenerate primer)

0.16 µl

5U/µl ExTaq

14.04 µl

H2O

2nd PCR cycle

95˚C, 1min
94˚C, 30 s -> 68˚C, 30 s -> 68˚C, 3 min 30 s „ª„­
94˚C, 30 s -> 68˚C, 30 s -> 68˚C, 3 min 30 s x 13
94˚C, 30 s -> 44˚C, 30 s -> 68˚C, 3 min 30 s „ª„®
68˚C, 5min
4˚C

3rd PCR reaction (total 20 µl)

1.00 µl

2nd PCR product (DF=50-200)

2.00 µl

10x ExTaq buffer

1.60 µl

2.5mM dNTP

0.60 µl

10 pmol/µl GSP3

0.60 µl

100 pmol/µl A1-3 (degenerate primer)

0.10 µl

5U/µl ExTaq

14.10 µl

H2O

3rd PCR reaction (the same as 2nd reaction)

94˚C, 1min->95C, 1min

95˚C, 1min
94˚C, 30 s -> 68˚C, 30 s -> 68˚C, 3 min 30 s „ª„­
94˚C, 30 s -> 68˚C, 30 s -> 68˚C, 3 min 30 s x 13
94˚C, 30 s -> 44˚C, 30 s -> 68˚C, 3 min 30 s „ª„®
68˚C, 5min
4˚C

-@ After 3rd PCR reaction, clone the longest product into a vector. We usually use pGEM T easy vector (Promega). Blue/White selection is effective for the cloning.

-@ It sometimes happens that monoprimer products carry the right extended clones, so do analysis without discarding them.

-@ To check whether the extended sequence by TAIL-PCR is correctly derived from the original sequence, it is necessary to do PCR with primers based on the obtained sequence.

-@ Temperature of extension during PCR cycle is at 68˚C rather than at 72˚C.@ This avoids depurination of DNA and helps to get longer PCR products.

@

(2) TAIL-PCR with KOD Dash DNA Polymerase

1st PCR reaction@ total 20 µl
1.0 µl 40 ng/ul genomic DNA
2.0 µl 10x KOD dash buffer
2.0 µl 2 mM dNTPs
0.4 µl 10 pmol/µl GSP1
1.0 µl 100 pmol/µl A1-3 (degenerate primer)
0.4 µl 2.5U/µl KOD Dash
13.2 µl H2O

1st PCR cycle
94˚C, 1 min¨95˚C, 1 min@@ (add primer at this step)
(94˚C, 20 sec->65˚C, 5 sec->74˚C, 30 sec) x 5
94˚C, 20 sec->30˚C, 30sec-> (1 or 3 min)¨74˚C,30 sec
94˚C, 20 sec->68˚C, 5 sec->74˚C, 30 sec@ „ª„ª„­
94˚C, 20 sec->68˚C, 5 sec->74˚C, 30 sec@@ x 13
94˚C, 20 sec->44˚C, 5 sec->74˚C, 30 sec@ „ª„ª„®
72˚C, 1 min
4C

2ndPCR reaction total 20 µl
1 µl 1st PCR products (DF = 50)
2 µl 10x KOD dash buffer
2 µl 2 mM dNTPs
0.4 µl 10 pmol/µl GSP2
0.8 µl 100 pmol/µl A1-3 (degenerate primer)
0.3 µl 2.5u/µl KOD Dash
13.5 µl H2O
2nd PCR condition
94˚C, 1 min->95˚C, 1 min@ @(add primer at this step)
94˚C, 20 sec->68˚C, 5 sec->74˚C, 30 sec „ª„ª„­
94˚C, 20 sec->68˚C, 5 sec->74˚C, 30 sec x 10
94˚C, 20 sec->44˚C, 5 sec->74˚C, 30 sec „ª„ª„®
74˚C, 1 min
4˚C

3rd PCR reaction total 20 µl
1 µl 3rd PCR products (DF = 50)
2 µl 10x KOD Dash buffer
2 µl 2 mM dNTPs
0.6 µl 10 pmol/µl GSP3
0.6 µl 100 pmol/µl A1-3 (degenerate primer)
0.2 µl 2.5 u/µl KOD Dash
13.56 µl H2O
3rd PCR cycle
94˚C, 1 min->95˚C, 1 min @(add primer at this step)
94˚C, 20 sec->68˚C, 5 sec->74˚C, 30 sec „ª„ª„­
94˚C, 20 sec->68˚C, 5 sec->74˚C, 30 sec x 9 or 10
94˚C, 20 sec->44˚C, 5 sec->74˚C, 30 sec „ª„ª„®
74˚C, 1 min
4˚C