Literatures for TAILiThermal asymmetric
interlacedj-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 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 GeneAmp
9600iPerkin-Elmerj 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.
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 |
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
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 „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
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 „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.
@
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