Biomedical Engineering Reference
In-Depth Information
3.2.1. Coamplification and Linkage of Two PCR Products
1.
Two hundred nanograms of male or female genomic DNA is made up to a final
volume of 50
µ
L containing 20 m
M
Tris-HCl (pH 8.4), 50 m
M
KCl, 2.5 m
M
MgCl
2
, 200
M
deoxynucleotide triphosphate (dNTP), 50 pmol of each primer
BIO5TSPY and NY3DPB1, 20 pmol of each primer A5DPB1HL and
A3TSPYHL, and 2.5 U of
Taq
polymerase.
µ
2.
PCR conditions: denaturation for 5 min at 94°C, 35 cycles of annealing for 90 s
at 55°C, extension for 90 s at 72°C, and denaturation for 45 s at 94°C.
3.
One microliter of this first PCR product is used as template in a second PCR:
Final volume of 50
L with the same buffer conditions as the first PCR, except
for 1.5 m
M
MgCl
2
and 25 pmol of primer NTPY5 and 25 pmol of primer
N2DPB1. PCR conditions: 94°C for 2 min, 30 cycles of 94°C for 45 s, 55°C for
1 min, 72°C for 3 min, and a final extension of 72°C for 5 min.
µ
4.
Analyze amplified DNA fragments by electrophoresis on 2% agarose gels and
stain with ethidium bromide.
3.2.2. Determination of a Lower Sensitivity Cutoff for the Co-Amplification
Linker PCR Procedure
In a separate line of experiments to determine a lower sensitivity cutoff for
the co-amplification linker PCR procedure using male genomic DNA, a third
PCR is conducted.
1.
Serial dilutions of male genomic DNA from 0.01 ng (corresponding to approx 1
cell) and to 5 ng (approx 500 cells) are used as templates.
2.
The second PCR is modified in such a way that to the 50-
µ
L volume of the first
PCR, 50
L of the second PCR mixture is added with a final amount of each of
the primers NTPY5 and N2DPB1 of 100 pmol and 5 U of
Taq
polymerase.
µ
3.
Five microliters of this second PCR product is used as template in the third PCR:
final volume of 50
µ
L containing 20 m
M
Tris-HCl (pH 8.4), 50 m
M
KCl, 1.5 m
M
M
dNTP, 40 pmol each of primers nesTSPY5 and nesDPB1-3,
and 2.5 U of
Taq
polymerase. PCR conditions: denaturation for 2 min at 95°C;
40 cycles of denaturation for 45 s at 95°C, annealing for 60 s at 55°C, extension
for 2 min at 72°C.
MgCl
2
, 200
µ
4.
Analyze amplified DNA fragments by electrophoresis on 2% agarose gels and
stain with ethidium bromide.
The two-step PCR procedure with co-amplification and linkage of the
TSPY
and
HLA-DPB1
PCR products followed by nested PCR in the traditional PCR
setup produce clear bands after gel electrophoresis corresponding to the
predicted length of the linked PCR product (531 bp) in only the male samples.
Dilutions of male genomic DNA have shown that a concentration down to
0.05 ng, corresponding to genomic DNA from approximately seven cells, pro-
duces a positive PCR signal.
