Molecular Methods for Diagnosis of Genetic Diseases
Evolving Molecular Methods for
Mohammad Al-Haggar, MD.
Professor of Genetics
- Mutations permanent changes in DNA in either
1. Germ line (gamete) all cells of an individual, OR
2. Somatic line only in % body cells.
- HGP 2003 basic molecular defect of most genetic
diseases phenotype-genotype correlations.
- Since 2003 methods for detection of mutation are
1. Old basic tedious and time consuming experiments.
2. Rapid and high throughput in a narrow spaced
• Some are used together in succession, Others are changing.
1. Southern blotting
2. Restriction fragment length polymorphism (RFLP)
3. PCR/RFLP (size analysis of PCR products)
4. Amplification-refractory mutation system (ARMS) PCR
5. Heteroduplex migration analysis
6. Single- strand conformational polymorphism (SSCP)
7. Denaturating gradient gel electrophoresis (DGGE)
8. Denaturating high performance liquid chromatography
• DNA digestion by restriction enzyme
restriction fragments (RFLP) of different size and
molecular weight based on recognition sites.
• Separation on agarose gel by electrophoresis.
• Denaturation of DNA fragments by soaking gel in
an alkaline solution.
• Transfer to nylon membrane.
• Hybridization complementary probes (radio-/
Amplification (PCR) RFLP
• Amplification of DNA stretch by PCR using
labeled nucleotides restriction fragments
• Recognition of mutant alleles especially in
• Use of multiple enzymes multiplex reaction
Amplification Refractory Mutation
• Amplification of specific allele used for
common mutations e.g. β-thalassemia.
• It consists of 2 complementary reactions and
utilizes 3 primers 1 constant (complement to
template in both reactions), the other 2 primers
differ at 3' terminal, 1 for wild and 1 for mutant
N M N M N M N M
Homozygous normal Homozygous mutant Heterozygous No Signal
• Screening for the presence or absence of
mutation in an amplified exon.
• Basis: single DNA strand folds to form a
complex 3-D structure maintained by intra-
molecular bonds. 80% of mutations
variable folding variable electrophoretic
• Differential mobility between test and control
DNA samples +ve mutation sequencing.
Heteroduplex migration analysis
• Differential migration of DNA heteroduplexes
compared to homoduplexes (2 mismatched
single strands) run on a polyacrylamide gel.
• Heteroduplex PCR products from a
suspected carrier are denatured allowed to
reanneal to a normal PCR product
• Useful for detection of carriers of X-linked
Denaturating gradient gel
• More demanding procedure than SSCP and
heteroduplex migration analyses with a higher
sensitivity (over 90%) for identifying mutations
especially in survey studies.
• Basis: normal and mutant sequences will show
different band patterns on a gel. The PCR
products are run on a special denaturing gradient
gel that contains increasing concentrations of a
denaturing chemical e.g. urea.
Denaturating high performance liquid
• Similar to DGGE, detects heteroduplexes owing to
their abnormal denaturing profiles.
• Basis: differential separation of mismatched
heteroduplexes which form after the reannealing
of normal and mutant DNA strands. PCR products
are injected in a column containing an increasing
gradient of mobile phase (acetonitrite) required
to elute each homo- or heteroduplex.
• It is an extremely sensitive method for detecting
base substitutions, small deletions and insertions.
• It is a faster technique that achieves a higher
throughput than DHPLC in detection of the
heteroduplexes using fluorescence
Oligonucleotide Ligation Assay (OLA)
• A pair of oligonucleotides designed to anneal
to adjacent sequences within a PCR product
If perfectly hybridized joined by a DNA
• Oligonucleotides complementary to normal
and mutant sequences are differentially
labeled and the products are identified by a
High-resolution melt curve analysis
• Fluorescent dyes intercalates with double strand
DNA (during PCR) PCR products heated
separation of the two strands.
• Fluorescence ↓, as DNA strands dissociate
melting profile depends on the PCR product size
• It is very sensitive. Its detection capability is
dependant upon fragment length, sequence,
mutation, PCR quality and analytical equipment.
• Quantitative PCR multiple hardware
platforms for real-time PCR, and fast versions
that can complete a PCR reaction in less than
DNA microarrays (DNA chips)
• Small devices (coated glass microscope slides) large
numbers of different DNA sequences placed on slide using
an automated robot.
• Gene Chips different Oligo DNA sequences produced by
in situ synthesis microscopic and submicroscopic VNTR
for the whole genome in a single assay.
• Major microarray platforms: for genomic DNA profiling;
1. Comparative genomic hybridization (CGH) arrays use a
two-color scheme infers VNTR in a test sample
compared to a reference sample .
2. Genotyping arrays No control sample; intensity of the
hybridization signal α DNA copies useful for SNPs.
• Direct determination of DNA sequences using
the new generations of automated
• Basis Amplification using fluorescent
nucleotides PCR products electrophoresis
fluorescence signal during its moves
through a window converted into an
electronic signal analysis by a computer
a color graph that signify each nucleotide.
• Nowadays fully automated (hardware
• Up to date revision on gene atlas is sometimes
necessary (discovery of new exons in a gene
• Basic molecular techniques are subjected to
modifications and optimizations based on:
1. Genetic disease,
2. Mutation type, and
3. Troubleshoots in lab.
• Fixed frames of laboratory technique:
3. Separation (differential migration),
4. Labeling, and