PCR-SSP
The polymerase chain reaction using sequence specific primers (PCR-SSP) is a
molecular typing technique that replicates genomic DNA extracted from intact
nucleated leucocytes in an anti-coagulated peripheral blood sample. The tissue typing
laboratory perform PCR-SSP as its main method of HLA typing on all initial entry
renal patients and potential donors, as well as patients referred by local consultants
with possible disease states linked to a specific HLA type, such as HLA-B27 linked
Ankylosing Sponylitis. The level of PCR resolution is medium, which defines the
alleles to two digits and equates to a serological equivalent. The laboratory uses a
serological method of typing as a control, and to confirm unusual or homozygous
PCR results.
Extracted DNA is mixed with PCR solution and Taq DNA polymerase in the volumes
shown in Table 1.
Table 1
Volumes of PCR pre-mixture
Plate
PCR solution
Class I
Class II
B locus
C locus
DQ locus
305.8l
108l
162l
60l
30l
DNA
1.0g/l)
196.5l
69l
104l
38.5l
19.5l
(~0.75- AmpliTaq ® DNA
polymerase
8.6l
2.9l
4.3l
1.6l
0.8l
PCR solution contains,






Magnesium chloride (MgCl2 ) - a cofactor for DNA polymerase
Cresol red - to allow visualisation of the solution
Glycerol - a density medium to aid loading of the solution onto a gel prior to
electrophoresis.
Deoxyribonucleotide triphosphates (dNTP’s), the basic components of DNA,
which enable the extension primers and complimentary regions of DNA to be
replicated.
PCR buffer - providing an optimal chemical environment for DNA
polymerase
Deionised H2O
Taq DNA polymerase is an enzyme isolated from the thermophilic bacterium
Thermus aquaticus. The bacterium normally thrives in hot springs and hydrothermal
vents. It has a half-life of 1.6 hours at 95ºC and is therefore able to survive the high
temperatures required for DNA amplification. The enzyme catalyses the addition of
dNTP’s complementary to the template strand in the presence of MgCl2. The
optimum temperature for the polymerase is 70ºC.
The mixture is added to wells in a plate containing primer pairs. Primers are short
strands of nucleic acid, which are complementary to the beginning and end of the
DNA fragment to be replicated. The primer pairs are chosen if known to be unique to
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a specific allele group. Control primers are included, designed to replicate a nonpolymorphic region of the human growth hormone gene in each reaction, to show that
amplification has been successful. The wells also contain chill out wax, which
solidifies below 10ºC securing the primer mix in the well, and acting as a vapour
barrier to prevent evaporation, while the red colour aids visualisation of the primer
mix.
Amplification of DNA takes place in a thermal cycler. The process involves a series
of up to thirty cycles consisting of three steps.
1) The double stranded DNA is heated to 95ºC breaking the hydrogen bonds
between them and separating the two strands.
2) As the temperature is reduced, the primers anneal to the denatured DNA
where they find a complementary sequence.
3) DNA polymerase catalyses the replication of DNA using the single strand as a
template, by anchoring to the complex, incorporating dNTP’s from the
solution and extending along the strand. During amplification DNA yield will
increase exponentially with each cycle.
The optimum temperature for the primers to anneal to a homologous sequence is
dependent on the ratio of A/T to C/G bases contained within the primer strand, and is
determined by the following equation where Tm is the melting temperature of the
primer. All primers are designed to anneal at approximately 60ºC.
Tm = 2(AT) + 4(GC)
At the end of the process the amplification products are analysed by electrophoresis,
which separates the DNA according to its molecular size. The contents of the plates
are loaded into wells in an agarose gel, which provides a solid but porous matrix. The
samples are held to the bottom of the well by the glycerol in the PCR mixture. The
negatively charged DNA moves through the gel towards the anode when an electric
current is applied. Smaller molecules will travel further through the gel. The gel
contains ethidium bromide, which binds to the DNA as it travels through the gel, and
will fluoresce under ultra-violet light. The gel is viewed by a digital imaging system
and a picture of the gel is taken (see CaseStudyworksheet.xls) which displays bands
of amplification product.
The control band with a molecular weight of approximately 800 base pairs should be
present in all lanes if the amplification process was successful and there was sufficient
DNA in the sample. Subsequent bands in a lane indicate a positive result for
amplification of a specific allele group, and if the distance travelled corresponds to the
molecular weight of the known amplification product, the result can be confirmed.
Positive bands are recorded on a sheet containing a list of known alleles (see
CaseStudyworksheet.xls). Due to the close proximity of loci on chromosome six,
certain HLA alleles tend to be inherited together limiting recombination. Examples
include B7 with Cw7, and DR1 with DQ5 (see Associations.xls). Certain groups of
antigens also share public epitopes. Using known antigen associations derived from
population studies as an aid to interpretation, the patient’s HLA type is deduced.
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