In-Cell Polymerase Chain Reaction - Clinical Applications of PCR - page 47

10. PCR buffer and reagents:

a. 20 m M Tris-HCl (pH 8.4) or 10 m M Tris-HCl (pH 8.8).
b. 50 m M KCl.
c. MgCl 2 .
d. Deoxynucleotide triphosphate (dNTP).

11. DNA sequencing reagents.

12. Thermal cycler.

13. Automated DNA sequencing equipment and agarose gel equipment.

3. Methods
3.1. Overview of the General Principle
The main steps in the procedure are outlined briefly here. A detailed
description follows in next sections. Figures 1 and 2 illustrate the general prin-
ciple of the in-cell linker PCR method. In the model system, CD71 + cells are
isolated from mixtures of male and female umbilical cord blood with use of
anti-CD71 monoclonal antibody-coated immunomagnetic particles. The dif-
ferent steps of the procedure, which include several changes of the buffer in
which the cells are suspended, can then be performed easily by immobilization
of the beads binding the cells by use of a magnet ( see Note 1 ). After fixation
and permeabilization, the cells are resuspended in PCR buffer with the primers
BIO5TSPY, A3TSPYHL, NY3DPB1, and A5DPB1HL. During the first PCR,
two PCR products will be amplified in male cells: a sequence of the gene TSPY
and a sequence of exon 2 of HLA-DPB1 ( Fig. 1 ). TSPY (CYS14) is a Y-chro-
mosome-specific, single-copy gene encoding a testis-specific protein ( 13 ) , and
was chosen as the male (“fetal”)-specific marker sequence ( see Note 2 ). The
HLA-DPB1 sequence was chosen as an example of a polymorphic gene of in-
terest because it is a well characterized gene and approx 80 alleles have been
reported so far (e.g., HLA Informatics Group, http://www.anthonynolan.org.uk
/HIG; [ 14 ] ); therefore, the probability that any two samples have some poly-
morphic differences in this sequence is large. Thus, HLA-DPB1 sequences from
either the male or female cells can easily be defined in most cases. At the end
of the first PCR, the TSPY and the HLA-DPB1 PCR products will begin to form
a linked PCR product because of complementary primer tail sequences ( Figs. 1
and 2 ). These tail sequences have no marked homology to any known human
genome sequences. Differences in primer concentrations added will partly
force this to happen. This first PCR and linkage of PCR products are supposed
to occur to a great extent inside the fixed male cells. In the fixed female cells,
only the HLA-DPB1 sequence will be amplified from the genomic DNA. After
the first PCR, the PCR mixture is removed from the cells, and the cells are
washed once in 1X PCR buffer and resuspended in the second PCR mixture
including the nested primers NTPY5 and N2DPB1 ( Fig. 1 ). Removal of the