Biomedical Engineering Reference
In-Depth Information
should be sequenced in both directions to generate a 'read pair' - two reads across the
same genomic region, but on opposite strands. Many downstream SNP discovery tools use
read pair information to assess the confidence of variant predictions. The disadvantage of
sequencing in both directions, of course, is the added expense of universal primer sequences.
However, this expense can be avoided by using a '10-to-1' protocol for sequencing, in which
one PCR primer serves as the sequencing primer when it is added at 10
×
concentration
(see Protocol 2.2).
PROTOCOL 2.2 PCR and Sequencing with the 10-to-1 Protocol
Equipment and reagents
10
×
PCR buffer (0.5
M
KCl, 0.1
M
Tris-HCl (pH 8.3), 35 m
M
MgCl
2
)
•
10
×
4dNTPs(4
×
1m
M
)
•
10
×
Primer 1
k
,
l
(10
×
μ
M
)
•
10
×
Primer 2
l
(1
μ
M
)
•
Hot-start Taq DNA polymerase (JumpStart Taq, Sigma-Aldrich or Platinum Taq,
Invitrogen Life Technologies)
•
BigDye version 3 mix (Applied Biosystems)
•
5
×
sequencing buffer (Applied Biosystems)
•
96- or 384-well purification plates (Princeton Separation or Genetix)
•
Column cleanup kit (Qiagen or GE Healthcare).
•
Method
PCR
1 Set up PCR amplifications in a 96- or 384-well format. For each target, set up a 10
μ
l
reaction containing 1
×
PCR buffer, 1
×
4dNTPs,1
×
Primer 1, 1
×
Primer 2, 4 ng of
DNA and 0.15 U of hot start Taq DNA polymerase.
2 Thermocycle as follows: 95
◦
C, 2 min; (92
◦
C, 10 s; 58
◦
C, 20 s; 68
◦
C, 30 s)
×
35; 68
◦
C,
10 min.
3 Verify a successful PCR by gel electrophoresis before sequencing.
m
Sequencing
4 For each PCR product, set up a 12
μ
l sequencing reaction containing 2.5
μ
l
of PCR reaction mixture, 2
μ
l of BigDye version 3 mix and 1.0
μ
lof5
×
sequencing
buffer.
5 Thermocycle as follows: 96
◦
C, 2 min; (96
◦
C, 15 s; 50
◦
C, 1 s; 60
◦
C, 4 min)
×
25.
6 Remove unincorporated dye terminators.
n
Load reactions in 96- or 384-well format onto
an ABI 3730XL for capillary electrophoresis (Applied Biosystems).
