PCR TECHNIQUES: COURSE CONTENT

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Molecular Biology: PCR techniques
Molecular Biology:
PCR Techniques
Author: Prof Estelle Venter
Licensed under a Creative Commons Attribution license.
(DIAGNOSTIC) APPLICATION OF PCR

To study minute quantities of DNA. From e.g. a single sperm cell, bloodstains, hair and bones of
murder victims. A fragment of up to 5 kb can be amplified and be used for a variety of purposes.

The amplification of inserts of bacterial plasmids with primers based on the flanking vector
sequence.

Amplification of any DNA from which the sequence is available. Databases of DNA sequences
have been established where millions of DNA sequences are freely available.

Designing primers on the basis of homologous sequence e.g. to isolate a gene from the
chimpanzee using known human primers. Degenerate primers, obtained from the sequence of the
translated protein, can also be used. (Degenerate primers have not been explained in this
course).

Study phylogeny and evolution. Sequencing data of the bacterial 16S rRNA and of the fast
evolving genomes of mitochondria and plant chloroplasts are used in this field of study. PCR
amplicons of these genes are sequenced and with the use of specific software programs
analyzed.

The use RFLP, PCR and sequencing to determine the specific serotype of e.g. a virus. This play
an important role in the epidemiology of diseases e.g. determining the source of an outbreak.
Pathogenesis of disease.

Amplification of RNA. RNA is first converted into single-stranded cDNA with the enzyme reverse
transcriptase and then used in a PCR. A useful application of RT-PCR is measuring the relative
amounts of mRNA in different tissues or in the same tissue at different times. This is mainly done
by real-time PCR.

By amplifying and sequencing of genes or using RAPD of RFLP techniques, genomes of different
organisms can be compared - Genotyping of microorganisms.

Genetic disorders can be identified by the identification and characterization of the gene
responsible for the disease.
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Molecular Biology: PCR techniques

PCR can be used in the diagnosis of cancer by the detection of mutation/s in oncogenes or
tumor-suppressor genes.

Typing of tissue at a DNA level and comparison between individuals e.g. for bone-marrow
transplantation.

Linkage analysis of genetic markers –A marker is based on a polymorphism in a population, the
existence of two or more alleles, or genetic variants. Markers can be phenotypic (genetic
diseases), on the protein level or mutations on the DNA level (point mutations, insertions and
deletions). A genetic map of a species indicates the location of genetic markers relative to each
other. Mutations in genes are genetic markers that in many cases influence the phenotype.
RFLP with Southern blotting were the first techniques to be used to identify these markers. PCRbased techniques, like microsatellites (use of short random primers), AFLP’s (amplified fragment
polymorphism) or SNP’s (single-nucleotide polymorphisms) are currently used. These variations
of the PCR are not discussed in this course).

A combination of mutations at different positions within a gene can be analyzed using PCR.

Forensic identification and paternity testing. By comparing genotypes of parents and offspring or
comparing DNA from a source to a specific individual, relationships can be revealed.

Gene expression. The traditional technique to detect the expression of a given gene in a given
tissue is the Northern blot analysis of mRNA. Amplification of cDNA is a rapid and sensitive
alternative provided that:
o
The amount of mRNA can be quantified, most conveniently by co-amplification of an
internal marker or the use of quantitative real-time PCR.
o
The appropriate controls are tested to check if the signal has been amplified from
contaminating chromosomal DNA.
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