Topic 3: Molecular techniques used in medical diagnosis

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Topic 3 Molecular techniques used in medical
diagnosis

• Goal for this topic
• gain a basic understanding of the molecular
  techniques that are used every day in the
  diagnostic lab and how those techniques help to
  dissect the cause of disease

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Separation of DNA Fragments by Gel
Electrophoresis
-
-

• Used to isolate/separate DNA fragments by size
• DNA is negatively charged- use a current to
  separate different sizes
• Visualize the DNA with dye that intercalates
  into strands

• Sequencing gel (acrylamide) -
• used to separate single base pairs

-

• Agarose gel - used to separate
• 10s-1000s of base pairs

• Pulsed field gel electrophoresis (agarose) -used
  to separate very large fragments

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Restriction Endonucleases
P
OH
Blunt cut
OH
P

• Endonucleases are derived from bacteria
• They cut INTERNALLY
• They recognize a specific PALINDROMIC sequence
• REMEMBER when you cleave a
• phosphodiester bond, you get a 3 OH and
• a 5 phosphate (P).

P
OH
OH
5 overhang
P
P
OH
5 overhang
OH
P
P
OH
3 overhang
OH
P
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Methods of Sequence Analysis
The human genome project utilized a shotgun
cloning approach - cloning down from very large
fragments to workable ones that are
easily sequenced.
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Big Dye Sequencing
ddATP
ddGTP
ddTTP
ddCTP
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Polymerase Chain Reaction
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PCR Analysis - Multiple rounds of amplification
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Lets look at an example of amplification.
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5 GGATTCGATCATTCGGGCCAATAGCATACTTAGGCCATACCGAGC
3 3 CCTAAGCTAGTAAGCCCGGTTATCGTATGAATCCGGTATGGCTC
G 5
mutation you want to look for
PRIMERS TO USE 5 ATTCGAT 3 5 TCGGTAT 3
QUESTIONS WHERE WOULD THE PRIMERS
HYBRIDIZE? HOW BIG WOULD YOURE AMPLIFIED
PRODUCT BE?
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Lets look at an example of amplification.
WHERE WOULD THEY HYBRIDIZE?
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5 GGATTCGATCATTCGGGCCAATAGCATACTTAGGCCATACCGAGC
3 3 CCTAAGCTAGTAAGCCCGGTTATCGTATGAATCCGGTATGGC
TCG 5
primer 2 - 3 TATGGCT 5
5 ATTCGAT 3 primer 1
HOW BIG WOULD YOURE AMPLIFIED PRODUCT
(AMPLICON) BE? 41 bp
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Polymorphisms in our genome can be used to look
for linked disease genes

• Types of polymorphisms
• single nucleotide polymorphisms (SNPs)
• variable number of tandem repeats (VNTRs)
• restriction fragment length polymorphism (RFLPs)
• These polymorphisms are inherited JUST LIKE OUR
  GENES!!!

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Linkage analysis The polymorphism can only be
used to predict the presence of the disease gene
if it is LINKED
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Patterns of Genetic Inheritance
RECESSIVE need 2 mutant alleles to see the
trait Example retinoblastoma
DOMINANT Only need one bad copy to see the
trait Example Marfans syndrome
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X-linked Inheritance

• most often affects males
• have female carriers

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RFLP Analysis
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Southern Blotting
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An example of VNTR linkage analysis

• Questions to answer
• 1) Does this trait appear to be inherited in a
  dominant or recessive fashion?
• 2) For each parent (generation II), which, if
  any, of their alleles appear to be linked to a
  mutant gene?
• 3) We are trying to determine if individual 3B is
  at risk for developing heart disease, as he is
  still quite young. What is his risk with respect
  to this locus?
• 4) Do you think that this VNTR will be helpful
  for ALL families with a history of heart disease?

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The use of PCR in Forensics Medicine
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Fragile X syndrome is caused by the expansion of
a VNTR region

• long, narrow face and prominent ears, jaw and
  forehead
• seizures
• mental impairment, ranging from learning
  disabilities to mental retardation
• ADD
• obsessive/compulsive
• 11200 males, 12500 females - most common
  inherited cause of learning disabilities
• VNTR is in the 5 UTR of the FMR1 gene -
  expansion causes lower expression of the gene

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DNA microarray analysis
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Preparation of cDNA from mRNA
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Topic 3 Molecular techniques used in medical
diagnosis

• Goal for this topic
• gain a basic understanding of the molecular
  techniques that are used every day in the
  diagnostic lab and how those techniques help to
  dissect the cause of disease

• restriction endonucleases
• sequencing
• PCR amplification
• VNTR and RFLP linkage analysis
• cDNA microarray analysis