In-Cell Polymerase Chain Reaction - Clinical Applications of PCR - page 52

3.5.1. PCR Co-Amplification and Linkage of the PCR Products
1.

Resuspend fixed and permeabilized cells (approx 12-15,000 cells from 1 mL
blood), derived from the CD71 + cell isolation procedure, in 50 L PCR mixture

containing 20 m M Tris-HCl (pH 8.4), 50 m M KCl, 2.5 m M MgCl 2 , 200

µ

M dNTP,
100 pmol of each primer BIO5TSPY and NY3DPB1, 40 pmol of each primer
A5DPB1HL and A3TSPYHL, and 5 U of Taq polymerase.

µ

2. Perform PCR under the following conditions: denaturation for 5 min at 94°C,

followed by 35 cycles of annealing 90 s at 55°C, extension for 90 s at 72°C, and
denaturation for 45 s at 94°C.

3.5.2. The First Nested PCR
1.

After the first PCR, place the tube in a magnet (MPC) and remove the superna-
tant. One hundred microliters of 1X PCR buffer was added for 1 min and
removed, followed by the addition of 50 µ L of the second PCR mixture: final

volume of 50 L with the same buffer conditions as the first PCR except for

2.5 m M MgCl 2 and 100 pmol each of primers NTPY5 and N2DPB1.

µ

2. PCR conditions: 95°C for 2 min, followed by 35 cycles of 94°C for 45 s, 55°C for

90 s, 72°C for 90 s.

L of supernatant from the first PCR
is used as template in a second PCR with 1.5 m M MgCl 2 , 20 pmol each of
N2DPB1 and 5NDPB1 ( see Table 1 ), and 2.5 U of Taq polymerase; same PCR
program. This is to obtain an HLA-DPB1 exon 2 sequence of the female
samples for later DNA sequencing. After the third PCR, the supernatant is
removed using the magnet and stored at -20°C.

For some of the pure female samples, 2 µ

3.5.3. The Second Nested PCR
1.

Resuspend the cells in 50 µ L 1X PCR buffer and place them at 100°C for 8 min.

2. Two or ten microliters of the cell lysates or supernatants are used as template for

the third nested PCR: total of 50 L of PCR mixture containing 10 m M Tris-HCl

(pH 8.8), 50 m M KCl, 1.5 m M MgCl 2 , 0.8

µ

M dNTP,
20 pmol each of primers nesTSPY5 and nesDPB1-3, and 2.5 U of Taq poly-
merase.

µ L/mL Nonidet P40, 200 µ

3. PCR conditions: denaturation for 2 min at 95°C, followed by 35 cycles of dena-

turation for 45 s at 95°C, annealing for 60 s at 55°C, and extension 2 min at 72°C.
Amplified DNA fragments are analysed by electrophoresis on 2% agarose gels
and stained with ethidium bromide.

Results from an in-cell linker PCR experiment performed on CD71 + cells
derived from a male and a female pregnancy and a 1:1 mixture of cord blood,
respectively, are shown in Fig. 3A . Only pure male or mixed samples result in
a linked and nested PCR product of the predicted size of 495 bp. Linked PCR
products are present in the PCR supernatant after the third PCR step in samples