In-Cell Polymerase Chain Reaction - Clinical Applications of PCR - page 53

Table 1
List of Primers for Polymerase Chain Reaction Amplification a
BIO5TSPY 5' biotin-GCC AAT GTT GTA TCC TTC TCA GTG 3'
A3TSPYHL 5' CGC CAT TAC CAG TTG GTC TGG TGT C A G TTT CTC
TGC CGC ATG CAG GAC A 3'
NY3DPB1 5' GAC CGC CGG CCC AAA GCC CTC ACT C 3'
A5DPB1HL 5' GAC ACC AGA CCA ACT GGT AAT GGC G A A TTA GAT
GAG AGT GGC GCC TCC GCT CAT 3'
NTPY5 5' CTT CGG CCT TTC TAG TGG AGA GGT G 3'
N2DPB1 5' CCG GCC CAA AGC CCT CAC TCA CCT C 3'
nesTSPY5 5' TCG GGA AAG TGT AAG TAA CTG ATG GGC AGC 3'
nesDPB1-3 5' AAG CCC TCA CTC ACC TCG GCG CTG CAG GGT 3'
5NDPB1 5' GAG AGT GGC GCC TCC GCT CAT GTC CGC 3'
Y1NY5 5' CAG TGT GAA ACG GGA GAA AAC AGT 3'
Y2NY3 5' GTT GTC CAG TTG CAC TTC GCT GCA 3'
BY5NES 5' CAT GAA CGC ATT CAT CGT GTG GTC 3'
Y3NESNY 5' CTG CGG GAA GCA AAC TGC AAT TCT T 3'
a The linker tail sequence in primers A3TSPYHL and A5DPB1HL is underlined.
containing fixed male cells ( Fig. 3A , lanes 5 and 6). There are no clear differ-
ences in the results of the third PCR based on supernatant or cell lysate, respec-
tively, from the second PCR. The linked and nested PCR products cannot be
visualized in ethidium bromide-stained gels after the second PCR. A third (and
a second nested) PCR is necessary. This is also the experience of other in-cell
PCR procedures ( 16 , 17 ) .
3.6. DNA Sequencing
DNA sequence PCR products with the use of automated DNA sequencing
according to the manufacturer's instructions. Here is Dye Primer (Cy-5' label-
ing) technology used (a Thermo Sequenase fluorescence-labeled primer cycle
sequencing method with 7-deaza-dGTP from Amersham Pharmacia Biotech,
UK). Sequencing primers: DPB1SEK5, Cy-5' GCG CCT CCG CTC ATG TCC
GCC CCC T 3', and TSPYSEK5, Cy-5' GTA AGT AAC TGA TGG GCA
GCT CGG CT 3'.
Direct DNA sequencing results (electropherograms) of the PCR products
are shown in Fig. 3B-D . Two HLA-DPB1 polymorphisms, which were found
to distinguish the male and female samples, are shown as examples. The DPB1
exon 2 polymorphisms are well described (HLA Informatics Group; http://
www.anthonynolan.org.uk/HIG) and are located respectively in codon 55 and
codon 56. The female sample has GAT at codon 55 and GAG at codon 56
( Fig. 3B ), whereas the male sample shows GCT at codon 55 and GCG at codon