Genetics Lecture No. 12: Detection Of Mutations & Genetic Variation
Monday February 25 , 2013
Individuals Differing Within A Species:
-Not all individuals within a species have identical genomes and it is necessary to know how to
distinguish between genetic variation, a wild type allele and a mutation? A polymorphic locus is
one with two or more alleles, where each allele is present in more than 1% of the populations.
The alleles of polymorphic loci are called genetic variations rather than wild types or mutants.
Advanced sequence technologies allow the sequencing of complete genomes from individuals
(e.g. single nucleotide substitutions and amino acid changes). Every individual within a species
has genetic changes which are unique to them and those that they share with others. For
example, most of the single nucleotide substitutions in the genomic region for the cystic fibrosis
gene do not have any effect on function (only about 2% of the nucleotides in this region are part
of coding sequences). The overall lengths of genomes differ as much as 1% between healthy
The Locus & Polymorphic Loci:
-The locus (plural loci) is any location within the genome that has a defined chromosomal
location, irrespective of any function (includes coding and non-coding regions, whether short or
long). Single base-pair substitutions, a single gene or several genes can be found at the locus,
while an allele describes the genetic variation present at a specific locus. Examples of
polymorphic loci include: Single Nucleotide Polymorphisms (SNPs or “snips”), insertions and
deletions (InDels or DIPs), Simple Sequence Repeats (SSRs), Copy Number Polymorphisms
(CNPs), and complex variants (none of the above). InDels or DIPs are a form of genetic
variation with short deletions and insertions (a type of SNP). A Copy Number Polymorphism
(CNP) or Copy Number Variant (CNV) is a category of genetic variation arising from large
regions of duplication or deletion, depending on the frequency of occurrence in a population.
Detecting Single Nucleotide Polymorphisms (SNPs) & Sickle Cell Anemia:
-SNPs are the most common type of genetic variation and can be spontaneous (arise during
DNA replication) or induced (usually by chemical mutagens) mutations. Essentially, SNPs are
base-pair substitutions. Snips are detectable as an SNP can create a restriction site
polymorphism at an EcoRI site, whereby the two SNP alleles will produce different-sized
restriction fragments. Southern blot analysis and hybridization with the probe can distinguish
among the three possible genotypes at a given SNP locus. A PCR analysis of the restriction
site-altered SNP alleles is then performed as the fragments are separated according to size
through gel electrophoresis. Finally, allele-specific oligonucleotide hybridization is necessary to
detect any SNPs that may be present.
-Sickle-cell anemia is a disease that arises by a single base-pair substitution (SNP) that results
in a single amino acids change in the β-globin chain of haemoglobin. The SNP promotes the
aggregation of haemoglobin under low oxygen conditions which distorts red blood cells into an unfavourable sickle shape. However, it is by this unnatural shape that heterozygotes are
resistant to malaria.
The Effect Of Temperature On Primers:
-In order to denature DNA strands, one has to increase the temperature and in order to allow
the annealing of both primers to the separated strands, one has to decrease the temperature (to
about 50-60°C). In primer sequences that are perfectly complementary to their target DNA
sequence, there is a greater annealing tolerance for the denaturing effect of increasing
temperatures than primers that are not quite a perfect match with their target sequence (which
anneal only at a low temperature). In PCR reactions, one can receive greater output of a target
sequence on the gel simply by increasing the annealing temperature, even if the same template
DNA and primers are used. Mispriming can result at lower temperatures where primers tend to
anneal to inappropriate target sequences more often.
The Effect Of Tem