LMP301H1 Lecture Notes - Lecture 22: Restriction Fragment Length Polymorphism, Capillary Electrophoresis, Southern Blot
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14 Sep 2016
School
Department
Course
Professor
LMP301
Introduction to the Biochemistry of Human Disease
Lecture 22 – Molecular Diagnostics
1949: discovery of restriction endonucleases, Southern, DNA sequencing, synthetic
oligonucleotides
1953: Watson and Crick, DNA double helix
1958: isolation of DNA polymerase
1960s: hybridization
1970s: some kind of blotting based on the picture
1980s: PCR and RFLP
Sickle cell anemia
o Detection by RFLP and Southern
o Southern is very labor-intensive and takes over a week to finish PCR to amplify DNA;
more starting material reduces time to 1 day
PCR disadvantages: based on known sequence, contamination risk, may miss
heterogeneous large insertion/deletion
Molecular diagnostics
o Used in diagnosis, monitoring, therapeutic, predictive… reduce morbidity and mortality
o A rapidly growing field
Sanger sequencing
o Using DNA polymerase to synthesize complementary strand to ssDNA
o Termination by 2’,3’-dideoxynucleotides
o Analyze fragment using gel or capillary electrophoresis
o Newer DNA sequencing now available
Hepatitis B
o Monitoring antiviral therapy by measuring viral load
o rtPCR: fluorescent probe with nearby counterdye probe – completed polymerization
separate of probes and emit fluorescence. Measure fluorescence as a quantitative
value of products.
Diseases
Name
Causes
Characteristics
Diagnosis
Treatments
Sickle cell anemia
AT mutation in β-globin
gene (autosomal
recessive)
(PCR)-RFLP and
Southern blot –
mutation
destroys a
restriction
enzyme
digestion site,
resulting in
larger fragment
Hereditary
hemochromatosis
HFE gene mutations
Multiplexed
PCR-RFLP –
genotyping
based on
electrophoresis