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MOLECULAR ASSAYS FOR
GENETIC TESTING
Human Genetic Testing
Human genetic testing serves four
main purposes:
a. Prenatal diagnosis
b. Newborn screening
c. Carrier (heterozygote) detection
d. Disease predisposition
KARYOTYPING
Karyotyping of fetal cells from
amniotic fluid or chorionic villi
–gross chromosomal defects,
trisomies etc
Trisomy 21 karyotype
I - Molecular Hybridization
For the detection of a specific DNA
sequence in a heterogeneous
mixture of DNA
(e.g. whole genomic DNA etc)
Probes
Labeled, single stranded DNA
complementary to the target DNA
The label may be radioactive (P32) or
non radioactive (fluorescent
chromophores or biotin)
General scheme for
hybridization assays
Labeled
probe
Heterogeneous mix
of DNA
Probe binds target
DNA
Probes
• Target DNA sequence known
Short oligonucleotide probes,15-30 bases,
complementary to part of the target sequence
• Protein sequence known
Several oligonucleotide probes constructed
using the genetic code, to locate the target
sequence
Hybridization- the flow chart
Denature target DNA
Immobilize on insoluble matrix
Expose to labeled probes
Allow time for hybridization
Wash away unbound probe
Detect for the presence of label
APPLICATION
Mr. and Mrs. are expecting their second child. They
know that sickle cell anemia runs in both of their
families. They want to know whether this child could
be affected. Neither they nor their 10-year-old daughter
have shown any symptoms of the disease. They
decide to have DNA tests to determine the status of the
fetus, as well as to find out whether they in fact are
carriers of the disease gene.
They send their samples to a DNA diagnostic facility
where their samples are analyzed using the allele
specific oligonucleotide (ASO) probe technique.
Allele specific oligonucleotide probe detection of bs-globin mutation
SICKLE CELL ANEMIA
AT mutation in the b-globin gene
converts glutamate  valine at position 6 of
the protein
Normal
CT CCT GAG GAG AAG TCT GC
GA GGA CTC CTC TTC AGA CG
Mutant
CT CCT GTG GAG AAG TCT GC
GA GGA CAC CTC TTC AGA CG
Allele specific oligonucleotide probe detection of bs-globin mutation
Two probes are designed
One complementary to the normal DNA sequence
CT CCT GAG GAG AAG TCT GC
GA GGA CTC CTC TTC AGA CG
Normal probe
The other complementary to the mutant DNA sequence
CT CCT GTG GAG AAG TCT GC
GA GGA CAC CTC TTC AGA CG
Mutant probe
Allele specific oligonucleotide probe detection of bs-globin mutation
Normal Mutant
Probe Probe
Homozygous normal
AA
Heterozygous carrier
AS
Homozygous mutant
SS
= PROBE HYBRIDIZES
= PROBE DOES NOT
HYBRIDIZE
Allele-specific PCR
Normal CTCCTGAGGAGAAGTCTGCNNNNNNNNNN
Mutant CTCCTGTGGAGAAGTCTGCNNNNNNNNNN
Template DNA +
dNTPs+MgCl2+Taq
Polymerase,
primers
If Normal (A) amplification takes place
If Mutant (T) amplification does not take place
Template DNA +
dNTPs+MgCl2+Taq
Polymerase,
primers
If Normal (A) amplification does not take place
If Mutant (T) amplification takes place
APPLICATION
A 32-year-old female presents to your clinic with
concerns over a recently detected right breast lump. A
biopsy is performed and reveals an intraductal
carcinoma. She is invited to participate in an
experimental study that is being carried out to help
direct future treatment protocols and define new drug
targets for breast cancer. The researcher explains to
her that DNA microarrays (DNA chips) will be used
to study the differences in the gene expression
profiles of tumor versus normal cells. After
considering all the pros and cons she gives her
informed consent and allows her tissue samples to be
used.
Microarrays/DNA chips
Microarrays/DNA chips
small, solid supports onto which DNA
sequences from thousands of different genes
are immobilized
• Expression analysis
• Genotyping
SNP analysis
Mutation detection
Expression analysis using
microarrays
Applications of DNA
microarrays
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Cell specific expression
Gene regulation
Tumor profiling
Genetic variation
Microbial strain identification
Drug testing
APPLICATION
Mr. and Mrs. JD are expecting their first child. Mr. JD’s uncle
had died of cystic fibrosis (CF) and they recently learnt that a
distant cousin of Mrs. JD has also been diagnosed with CF.
They are worried that they might be carriers for the disease.
Their doctor suggests an amniocentesis to detect if their unborn
child has CF or is a carrier. They feel that an amniocentesis is
an invasive and risky procedure and decide that they first want
to be tested themselves to see if they are carriers for the
disease. If they learn that they both are carriers, they would like
to go through with the amniocentesis to see if their child is
affected.
The most common mutation, accounting for about 75% of CF
cases, is called delta F508 and can be screened using the
AFLP (amplified fragment length polymorphism)
technique.
• Human genomic DNA contains restriction
sites for restriction enzymes
• Very often a mutation can result in the
creation of destruction of a restriction site
e .g. GAATTC = restriction site for EcoRI
Destruction of restriction site
GAATTC GATTTC
or
Creation of restriction site
GGATTC GAATTC
RFLPS typed using PCR
Forward primer
MspI
Reverse primer
PCR Amplification
Restriction digestion with MspI
(Δ508F lead to loss of MspI site)
Run on agarose gel
400 bp
Forward primer
Reverse primer
300 bp
100 bp
APPLICATION
Mr. and Mrs. SZ just had their first child. The
phenylketonuria (PKU) blood test performed at birth
indicated a high level of phenylalanine in the blood.
The physician suggests a follow-up DNA test
immediately to confirm the PKU diagnosis. None of
the mutations known to cause PKU in the
phenyalanine hydroxylase gene is picked up by the
standard testing methods. The lab therefore decides
to carry out DNA sequencing of the child’s sample
to check for the presence of a novel gene mutation
DNA sequencing
Characterization of DNA sequence is
through dideoxy DNA sequencing
(Sanger method)
Automated
DNA
sequencing
A DNA sequence
APPLICATION
The body of an unidentified young woman is
found stuffed in a sack in a forest. She has
multiple stab wounds and her face has been
mutilated beyond recognition.
The parents of a girl, who had reported their
daughter missing a few days ago, are asked to
provide blood samples for DNA Profiling to
establish if the body may be of their daughter.
The sack is which the girl was found is found to
have several hair on it which do not belong to
the girl. They are collected as forensic
evidence.
DNA Fingerprinting by RFLPs
(1987-mid 1990s)
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DNA Fingerprinting by RFLPs (Restriction Fragment
Length Polymorphisms) was developed in the early
1980s by Sir Alec Jeffreys
It made use of genetic variation in the distance
between restriction enzyme sites
- Due to the presence of VNTRs (Variable Number
of Tandem Repeats)

Power of discrimination was in the range of 106-108
for a six probe analysis
Variable Number of tandem
repeats/ Short tandem repeats
(STRs)
Individual 1
7 repeats
Individual 2
8 repeats
Individual 3
9 repeats
DNA Fingerprinting
2
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1
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restriction
fragments
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Person 2
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Person 1
The Steps: DNA Fingerprinting
DNA prepared (from crime scene samples and suspects)
Cut with restriction enzymes
Restriction fragments separated
using gel electrophoresis
Transferred to a nylon membrane
Exposed to radiolabelled probe that
binds to its complimentary DNA
fragments
Photographic image obtained
The Colin Pitchfork Case
FIRST EXONERATION AND CONVICTION
BASED ON DNA EVIDENCE
 Two young women were raped and
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murdered in Narborough, England
(1983 and then in 1987)
Police contacted Alec Jefferys for
DNA fingerprinting
The first suspect (who had confessed) was excluded
5,000 local men were then asked to provide blood/
saliva samples
CP convicted in 1988
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Human Identity Testing
Crime scene investigation -- matching
suspect with evidence
Paternity testing -- identifying father
Missing persons investigations -whose body
Mass disasters -- putting pieces back
together
Inheritance Claims – who gets the
money
Historical investigations
Military DNA “dog tag”
DNA profiling requires a
reference sample
A DNA profile on its own has NO context
DNA profiling works by comparison
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Crime scene evidence compared to suspect (forensics)
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Soldier’s remains compared to a reference sample
(Armed Forces ID)
Child compared to alleged father (paternity)
Victim’s remains compared to a biological relative (mass
disaster ID)
Sources of Biological
Evidence
 Blood
 Semen
 Saliva
 Urine
 Hair
 Teeth
 Bone
 Tissue
DNA fingerprinting by RFLPs:
the downside
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RFLP testing requires a relatively large
amount of HMW DNA (50-250ng =
thousands of cells)
Not ideal for forensic evidence, in which
small, degraded samples are common
PCR to the Rescue!!
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Polymerase Chain Reaction = Molecular Xeroxing

Series of cycles of three successive steps, carried
out in a Thermal Cycler, “amplify” the desired DNA
fragment(s)
5 cycles of PCR = 64 copies of DNA
40 cycles of PCR = 1.099 x 1012 copies of DNA!!
The Steps: STR Typing
Extract and purify DNA
(from crime scene samples and suspects)
Carry out PCR
Run PCR product on a genetic analyzer
Assign genotypes
Singleplex PCR
Forward primer
DNA +
Reverse primer
+ dNTPs+MgCl2+Taq Polymerase
Multiplex PCR
Locus 1
Locus 2
DNA +
Primers
Locus 3
Locus 4
+ dNTPs+MgCl2+Taq Polymerase
Simultaneous
amplification of four
locations on a DNA
template
ABI 310
Analyzer
DNAGenetic
sequencer
separates amplified DNA
ABI 310 Genetic Analyzer:
Capillary Electrophoresis
 Amplified STR DNA
injected
 Electric current
applied
 DNA pulled towards
the positive electrode
 DNA separated out
by size:
 Large STRs travel
slower
 Small STRs travel
faster
 Color of STR detected
and recorded as it passes
the detector
Detector
Window
PATERNITY TESTING
FATHER
MOTHER
GIRL
Advantages of STR Typing
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< 1ng of DNA is required to type 13-15 STR
loci
Can be processed within 24 hrs
Relatively degraded DNA samples can be
used
Power of discrimination ranges from
1014-1023. World population is 6 x 109
APPLICATION
• A 60 year old heroine addict presents at the
OPD with a history of repeated episodes of
flu, fever, malaise, and maculopapular rash.
He reveals that has been sharing needles
indiscrinately with other addicts.
Laboratory investigations include ELISA for
HIV.
Enzyme-linked Immunobsorbent
Assay (ELISA)
• The ELISA method is a diagnostic test used to detect
antibodies or proteins associated with a specific clinical
conditions.
• The basic principle of an ELISA is to use an enzyme to
detect the binding of antigen (Ag) antibody (Ab).
• The enzyme converts a colorless substrate (chromogen)
to a colored product, indicating the presence of Ag:Ab
binding.
• An ELISA can be used to detect either the presence of
Ags or Abs in a sample, depending on how the test is
designed.
• The development of color in an ELISA test indicates a
positive result.
Coat plate with
capture mAb (in this case specific for
HIV antigens)
Add test samples and
standard
Add biotinylated (labelled)
detection mAb
Add
streptavidin-enzyme
Add chromogenic substrate for color
development
Indirect ELISA
Enzyme-linked immunobsorbent assay (ELISA)
Colorless
substrate
Colored
product
Analysis of gene expression
Northern blotting
mRNAs (rather than DNA) are
isolated, electrophoresed, blotted on
a membrane and hybridized using a
cDNA labeled probe
Western blotting
Proteins (rather than DNA or RNA) are
isolated, electrophoresed, blotted on
a membrane and hybridized using a
labeled antibodies
THE END!