5. Applications of PCR Flashcards
(21 cards)
applications of PCR
diagnostics
DNA fingerprinting
Diagnostics is used in
in medicine, plant pathology, environmental studies (source/tracking)
E.g. Avian sexing, genetic diseases (sickle cell trait,
cystic fibrosis, hemochromatosis, galactosaemia etc.
DNA fingerprinting is used in
Identifying Individuals (Paternity Testing, Forensics)
Identifying Populations/Population Studies
Identifying Species
DIAGNOSTICS: IDENTIFICATION OF PATHOGENIC
E. COLI STRAINS
Several strains of E. coli cause diarrhea and it is important to
differentiate them from non-pathogenic flora
Multiplex PCR can be used to differentiate among pathogenic
strains and non pathogenic strains
WHAT ARE DNA FINGERPRINTS?
Molecular (DNA) markers that can be used to identify individuals, populations or species
Perfect DNA Fingerprinting System
Large number of variable, genome wide markers
Reliable and highly polymorphic
Easy to generate & interpret
A DNA polymorphism is
a sequence difference compared to a reference standard that is present in at least 1–2% of a population
Polymorphic = a gene with more than one allele
Monomorphic = gene with only one allele
Polymorphisms may or may not have phenotypic effects
Polymorphic
A gene with more than one allele
Monomorphic
gene with only one allele
Polymorphisms may or may not have
phenotypic effects
To identify individuals markers should be
polymorphic and
hypervariable
hypervariable
should be unique in each individual
Recall the human genome has a lot of tandemly repeated DNA or satellite sequences
2 main types of satellite sequences:
Microsatellites or STRs or SSRs – up to 13bp long
Minisatellites or VNTR – 10-100 bp repeats but hypervariable
VNTRs have repeats – 10-25 bp
Microsatellites are usually the markers of choice to identify
individuals as
they are numerous (over 10,000 in the human
genome) and they are hypervariable
STR are
STR are repeats of nucleotide sequences
Different alleles contain different numbers of
repeats
STR genotypes are analyzed using
gel or capillary gel electrophoresis
The FBI has chosen a set of
13 STR for human identity applications
PLUS Amel, an additional STR used to determine sex
STRS – PATERNITY TESTING
15-20 STRs used usually 16
Each STR is named & is highly polymorphic
Each polymorphism (amplicon) is numbered
An alleged father can be excluded as the biological father with as little as few as
two to three mismatches
PATERNITY TEST — INDEX AND PROBABILITY
Each match receives a Paternity Index (PI) value.
The paternity index measures the strength of a particular match based on the relative uniqueness of the match
The calculation of this PI is dependent on the ethnicity of the alleged father as it is determined using allelic frequencies obtained from population data.
High PI = rare
Low PI = relatively common in population
For an alleged father to be the child’s father the CPI value must be
at least 100
A CPI value of 100 corresponds to a probability of paternity of 99.0000%
A CPI value of 10,000 translates to a probability of 99.9900% and so on
