1.

Why do you think that most laboratories were not aware of the problem of selective loss
of the GC B and HLA DQA1 4.1 alleles caused by post-amplification primer extension of
denatured products (as described by Grow et al.)? (2 points)
In the following article Grow et al. noticed at a training workshop a selective loss of GC
B and HLA DQA1 4.1 alleles. It was observed that the signal loss was related to the
transfer time between denaturation and analysis, which increased cooling time causes the
primers to extend. It is difficult for most laboratories to notice a loss of signal because
most of the time the genotype of the sample is unknown and the analyst are following
standard operating procedures.
2. Describe three parameters that can have an effect on whether the appearance of a weak
1.1 signal in a single source sample occurs (despite the absence of such an allele in the
individual concerned). (3 points)
In the article Crouse et al. a variety of parameters were seen to have an effect of the
appearance of a weak 1.1 signal in a single source sample. One parameter that had an
affect during typing when increased concentrations of DNA, greater than 5ng, were
presents it was likely for a weak 1.1 signal to occur. Also, if the typing strip was allowed
to develop over a period of time the weak 1.1 signal would be present. Lastly, if an
individual has 100% homology with DX 2 genotype it is more likely for the weak
1.1 signal to occur.
3. Define what is meant by the term genetic diversity. Compare and contrast the power of
discrimination (PD) for the HLADQA1 system in the Caucasian population (i) with and
(ii) without, the sub-typing of the 4 allele. Use the manufacturer's data for these
calculations. (6 points)
Genetic diversity is the variability of the genes within a species, which within a
population no two alleles are identical. This is important because the diversity allows for
a population to adapt to a change in environment and alleles that are more suited to that
environment will get passed along to off-springs.
4. As a scientist with Haplotype Inc., a biotech company specializing in the development of
human identity assays, you have been asked to prepare a primer extension PCR assay for
a few select SNP markers. The first step is to prepare immobilized oligonucleotide probes
bound to a nylon membrane in a reverse dot blot-type format. How would you go about
doing this and optimizing the parameters involved? (4 points)
First, the probe needs to be complimentary to the sequence of interest with a long
deoxyribothymidine homopolymer tail, poly(dT), in order to bind to the nylon membrane.
Immobilization of these probes can be performed under UV irradiation at 254 nm to
crosslink the thiamine to the nylon membrane. The parameters can be optimized by
increased UV exposure time, tail length, concentration of the probes, and hybridization
time.

5. (a) Which of the common HLA DQA1 alleles detected by means of the commercial kit
appeared
most
recently
in
our
evolutionary
history?
(b) What evidence can be adduced to support the hypothesis that the extensive
polymorphism at the HLA DQA1 locus was generated prior to speciation of Homo
sapiens? (3 points)
a. DQA2 is the most recently evolved.
b. In the following article provides evidence that HLA DQA1 locus was generated
prior to speciation of homo sapiens because Pan sp. and Gorilla gorilla both share
the same DQa1 locus with Homo sapiens. This was determined because they were
all able to be amplified by the use of the same primer, shared sequence homology,
and have similar alleles.
6. Provide two explanations whereby a single source sample can provide unequal dot
intensities in one or more loci of the PM system. (2 points)
Two explanations whereby a single source sample can provide unequal dot intensities in
one or more loci of the PM systems would be in a rape case where the victims DNA
cannot be separated from the offenders sperm. Another, explanation could be when there
is too much DNA cross hybridization which can give a false positive result.