Literature
for TAILiThermal asymmetric interlacedj-PCR
Liu, Y.G.
and Whittier, 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 Whittier, 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 polymeraseiTAKARA,
Kyotoj, 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 a GeneAmp 9600iPerkin-Elmerj
thermal cycler.@ Different PCR cyclers
may require optimization of the PCR conditions.
arbitrary primer
gene specific primer
Design a GSP
with Tm greater than 72C.
More than
50bp must remain downstream of a third gene- specific primer to check if the
TAIL PCR fragment obtained is correctly extended from an original sequence.
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 |
Transfer
the PCR plate from ice after the temperature of PCR machine 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@ Ş
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@ ŞŽ
68˚C, 5min
4˚C
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 |
|
95˚C,
1min
94˚C, 30 s -> 68˚C, 30 s -> 68˚C, 3 min 30 s Ş
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 ŞŽ
68˚C, 5min
4˚C
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 |
94˚C,
1min->95C, 1min
95˚C,
1min
94˚C, 30 s -> 68˚C, 30 s -> 68˚C, 3 min 30 s Ş
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 ŞŽ
68˚C, 5min
4˚C
-@ After the 3rd PCR
reaction, clone the longest product into a vector. We usually use the 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 the analysis
without discarding them.
-@ To check whether
the sequence extended 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.
@
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@ ŞŞ
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@ ŞŞŽ
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 ŞŞ
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 ŞŞŽ
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 ŞŞ
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 ŞŞŽ
74˚C, 1 min
4˚C