genetic testing family secrets copyright © 2009, university of rochester
TRANSCRIPT
Genetic Testing
Family Secrets
Copyright © 2009, University of Rochester
Genetic disease and testing
• The patient’s journey…
• Types of genetic tests
• Techniques used in genetic testing
• Implications of genetic testing
• The future…
Inherited disease
Inherited disease
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x23x23x23x23x23x23
Genetic disease
Symptoms
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Predictive testing Tells a person if she carries a mutation that will cause, or put her at higher risk for, a disease later in life.
Newborn screening Detects common disorders in newborns, where immediate treatment can prevent dangerous symptoms
Carrier testing Tells a person whether or not he carries a mutation that could be passed on to his offspring. One can be a carrier, but not be at risk for a disease (as in recessive genes)
? ? ? ?
Genetic Testing
Predictive testing Tells a person if she carries a mutation that will cause, or put her at higher risk for, a disease later in life.
Newborn screening Detects common disorders in newborns, where immediate treatment can prevent dangerous symptoms
Carrier testing Tells a person whether or not he carries a mutation that could be passed on to his offspring. One can be a carrier, but not be at risk for a disease (as in recessive genes)
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Genetic Testing
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Physician
GeneticCounselor
Test family members with disease symptoms?
Test patient?
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Treatment
Prevention
Reproduction
PhysicianGenetic Counselor
Genetic Testing:Molecular Techniques
DNA
RNA
Protein
Function
DNA
RNA
Protein
Function
Genetic testing
DNA
Protein
ProteinFunction
Levels of Genetic Testing
normal mutated
Analysis of whole chromosomes – for large changes; extra chromosome, very large deletions or insertions
atcgatcgatcg atcgaAcgatcgAnalysis of sequence – for small changes; mutations in the sequence, small deletions or insertions
Analysis of protein shape – for any change that may affect the folding of the protein
Analysis of protein function – if the functional protein is supposed to make something, then some tests can detect the presence or absence of the product
X X
DNA
Protein
ProteinFunction
normal mutated
Analysis of whole chromosomes – for large changes; extra chromosome, very large deletions or insertions
atcgatcgatcg atcgaAcgatcgAnalysis of sequence – for small changes; mutations in the sequence, small deletions or insertions
Analysis of protein shape – for any change that may affect the folding of the protein
Analysis of protein function – if the functional protein is supposed to make something, then some tests can detect the presence or absence of the product
X X
Levels of Genetic Testing
Examples of Mutations in the DNA Sequence…
aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc
Normal
Examples of Mutations in the DNA Sequence…
aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgataatcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc
Single base pair mutation
Examples of Mutations in the DNA Sequence…
(Sickle cell anemia)
aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc
Deletion
Examples of Mutations in the DNA Sequence…Examples of Mutations in the DNA Sequence…
(Cystic fibrosis)
aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc
Deletion
Examples of Mutations in the DNA Sequence…
(Duchenne muscular dystrophy)
aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaaaaaaaaaaaaaaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc
Insertion
Examples of Mutations in the DNA Sequence…
(Huntington’s disease)
aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatggtcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatggggatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacatttttttttaaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc
Multiple mutations
Examples of Mutations in the DNA Sequence…
(Diabetes, susceptibility to breast cancer)
aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatggtcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatggggatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacatttttttttaaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc
Multiple mutations
Examples of Mutations in the DNA Sequence…
(Diabetes, susceptibility to breast cancer)
How do we “look” at the DNA sequence?
• Sequencing
• RFLP analysis
• Probes– Dot blot– Microarray
• Gel electrophoresis • PCR
How do we “look” at the DNA sequence?
• Sequencing
• RFLP analysis
• Probes– Dot blot– Microarray
• Gel electrophoresis• PCR
Gel ElectrophoresisSeparates DNA fragments on the basis of size
Large fragments take longer than small fragments to migrate
through an agarose gel
How do we “look” at the DNA sequence?
• Sequencing
• RFLP analysis
• Probes– Southern blot– Dot blot– Microarray
•Gel electrophoresis•PCR
PCR (Polymerase Chain Reaction)
aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgatttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta
PCR
aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat
ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta
Melting
94°C
PCR
aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat
ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta
Annealing
58°C
cgggcat
aactagg
PCR
aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat
ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta
Elongation
72°C
cgggcat
aactaggaatcgaatgtgcccgtacgattcgatgcga
gctaagctacgctttgatcctcgggatagcta
PCR
aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat
ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta
Melting
94°C
cgggcat
aactaggaatcgaatgtgcccgtacgattcgatgcga
gctaagctacgctttgatcctcgggatagcta
PCR
aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat
ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta
Annealing
58°C
cgggcat
aactaggaatcgaatgtgcccgtacgattcgatgcga
gctaagctacgctttgatcctcgggatagcta
cgggcat
cgggcat
aactagg
aactagg
PCR
aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat
ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta
Elongation
72°C
cgggcat
aactaggaatcgaatgtgcccgtacgattcgatgcga
gctaagctacgctttgatcctcgggatagcta
cgggcat
cgggcat
aactagg
aactagg
gcccgtacgattcgatgcga
gctaagctacgctttgatcctcgggatagcta
aatcgaatgtgcccgtacgattcgatgcga
gctaagctacgctttgatcc
PCRMakes a lot of copies of a specific portion of DNA
PCR test for deletion
Electrophoresis
How do we “look” at the DNA sequence?
• Sequencing
• RFLP analysis
• Probes– Southern blot– Dot blot– Microarray
•PCR•Gel electrophoresis
Sequencing
aaaaaaaatgatgatgatgatgatgatgatgatgatgatgatg
“Known gene sequence:”
*******************************************
Patient DNA sequence
How do we find out if the patient’s sequence is different?
Sequencing
*******************************************ttttttttactactactac*
tttttttt
tttttttt
tttttttt
*******************************************
*******************************************
*******************************************
*******************************************tttttttt
actac*
actactactactag*
actactactactacgactact*
actactactacta*
Sequencing
*******************************************tttttttta*
*******************************************
*******************************************
*******************************************
*******************************************
ttttttttactac*
ttttttttact*
ttttttttac*
ttttttttacta*
Sequencing
*******************************************tttttttta*
*******************************************
*******************************************
*******************************************
*******************************************
ttttttttactac*
ttttttttact*
ttttttttac*
ttttttttacta*
Sequencing
*******************************************tttttttta*
*******************************************
*******************************************
*******************************************
*******************************************
ttttttttactac*
ttttttttact*
ttttttttac*
ttttttttacta*
Sequencing
*******************************************tttttttta*
*******************************************
*******************************************
*******************************************
*******************************************
ttttttttactac*
ttttttttact*
ttttttttac*
ttttttttacta*
Sequencing
*******************************************tttttttta*
*******************************************
*******************************************
*******************************************
*******************************************
ttttttttactac*
ttttttttact*
ttttttttac*
ttttttttacta*
a
Sequencing
*******************************************tttttttta*
*******************************************
*******************************************
*******************************************
*******************************************
ttttttttactac*
ttttttttact*
ttttttttac*
ttttttttacta*
a c
Sequencing
*******************************************tttttttta*
*******************************************
*******************************************
*******************************************
*******************************************
ttttttttactac*
ttttttttact*
ttttttttac*
ttttttttacta*
ta c
Sequencing
*******************************************tttttttta*
*******************************************
*******************************************
*******************************************
*******************************************
ttttttttactac*
ttttttttact*
ttttttttac*
ttttttttacta*
ta c a
Sequencing
*******************************************tttttttta*
*******************************************
*******************************************
*******************************************
*******************************************
ttttttttactac*
ttttttttact*
ttttttttac*
ttttttttacta*
ta c a c…
Sequencing
actactactactagtactactactactactactac*******************************************
aaaaaaaatgatgatgatgatgatgatgatgatgatgatgatg“Known gene sequence:”
Sequencing
actactactactagtactactactactactactacaaaaaaaatgatgatgatgatcatgatgatgatgatgatgatg
aaaaaaaatgatgatgatgatgatgatgatgatgatgatgatg“Known gene sequence:”
Mutation
Issues with Sequencing
• Informative• Time consuming
• Expensive
How do we “look” at the DNA sequence?
• Sequencing
• RFLP analysis
• Probes– Southern blot– Dot blot– Microarray
•PCR•Gel electrophoresis
RFLP Analysis
RFLP (Restriction Fragment Length Polymorphism) analysis relies on the use of restriction enzymes
These enzymes recognize specific DNA sequences (usually palindromes) and cut them…
RFLP AnalysisRestriction Fragment Length Polymorphism means that there are polymorphisms (differences) between people in the number of restriction sites (and therefore the length of the cut fragments). This person has 4 fragments after restriction digest.
This person has a mutation that eliminates one of the sites that the restriction enzyme cuts at. Therefore, this person has 3 bands, one of them being much larger than the rest. If this mutation was associated with a disease, a restriction digest would show that a carrier of the mutation had 3 fragments instead of 4
RFLP Analysis
RFLP Analysis
Genomic DNA is very long and contains a lot of restriction enzyme cut sites
PCR makes many copies of a small region of DNA, better for RFLP analysis
RFLP Analysis
RFLP Analysis
RFLP Analysis
Issues with RFLP analysis
• Can not detect all mutations – the mutation has to coincide with a restriction enzyme cut site
How do we “look” at the DNA sequence?
• Sequencing
• RFLP analysis
• Probes– Dot blot– Microarray
•PCR•Gel electrophoresis
Probes
DNA bases pair with complementary bases
Probes
Probe
A probe is an a short fragment of DNA that is complementary to part of a longer DNA sequence.
DNA strands can be separated with high heat.
As the temperature is lowered, smaller fragments that have complementary sequences (probes) will base pair faster than the long original strands of DNA
ProbesThe ability of a probe to bind depends on:
*Its complementarity to the DNA strand it’s binding to…
-Single base pair differences can affect binding depending on…
*The stringency of the binding conditions
-Temperature
-Buffer conditions
Low stringency
High stringency
How do we “look” at the DNA sequence?
• Sequencing
• RFLP analysis
• Probes– Dot blot– Microarray
•PCR•Gel electrophoresis
Dot Blot(A Southern blot without the gel…)
A patient’s DNA (genomic DNA or PCR products) is denatured and spotted to a membrane…
Dot Blot
Membrane is washed in a solution that contains radioactively labeled probe
Probe binds to complementary sequence
Dot Blot
Membrane is washed in a solution that contains radioactively labeled probe
Excess probe is washed off – only probe bound to DNA on the membrane remains
Probe binds to complementary sequence
Dot Blot
The membrane is exposed to autoradiography film.. Therefore, wherever there is radioactive probe, the film will be exposed…
Dot Blot
The membrane is exposed to autoradiography film.. Therefore, wherever there is radioactive probe, the film will be exposed…
And a black dot will be developed where the probe was…
Dot blot to diagnose cystic fibrosis (CF)
Normal
Normal probe
Mutant probe
CF patient (homozygous mutant)
CF carrier (heterozygous mutant)
DNA from a patient is spotted out twice, one will be used with a probe complementary to the normal sequence, the other will be used with a probe that is complementary to a mutated sequence.
Since a heterozygote has one copy of the normal gene and one copy of the abnormal gene, both probes can bind, but only half as much binds, making the dot lighter.
Issues with dot blots
• Can be used to test patient for multiple mutations
• Single base pair mutations may be hard to detect with dot blots
How do we “look” at the DNA sequence?
• Sequencing
• RFLP analysis
• Probes– Dot blot– Microarray
•PCR•Gel electrophoresis
Microarray(High throughput dot blots. Allows for testing of many different mutations…)
Microarrays start with a “chip” on which is spotted many different probes for different mutations. Each dot represents a different probe.
If this were a dot blot, it would be called a “reverse dot blot” because the PROBE is spotted, rather than the DNA.
Microarray(High throughput dot blots. Allows for testing of many different mutations…)
The chip is treated much like a southern or a dot blot, except it is washed with a patient’s DNA (fragmented with restriction enzymes, denatured and labeled with a flourescent dye), and it automated.
Microarray
Binds to a probe for a CF mutation
Binds to a probe for a colon-cancer susceptibility mutation
Binds to a probe for a breast-cancer susceptibility mutation
If a patient’s DNA binds to spotted probes, a computer detects this by the measuring the intensity of the flourescence emanating from that spot. A homozygote for a CF mutation will have a more intense spot than a heterozygote for the same mutation
Issues with Microarrays
• Expensive
Available Predictive Tests
Cystic Fibrosis
Tay Sachs Disease
Lou Gherig’s Disease (ALS)
Huntington’s Disease
Catastrophically high cholesterol
Rare Cancers
Inherited susceptibilty to cancerBreast Cancer Colon Cancer Thyroid Cancer
Benefits of Genetic Testing
If Negative
Relief
Fewer health check-ups and tests that go with being in a family that tends to have a higher risk of a particular genetic diseases
If Positive
Able to make informed decisions
May be possible to reduce the risk of coming down with severe symptoms
Limitations of Genetic Testing
• Mutations may not always lead to disease• Existing tests only look for the most
common mutations, some disease causing mutations will not be detected by conventional tests
• Small chance of errors in testing procedure
• Testing is not always matched by treatment
Limitations of Genetic Testing
• Mutations may not always lead to disease• Existing tests only look for the most
common mutations, some disease causing mutations will not be detected by conventional tests
• Small chance of errors in testing procedure
• Testing is not always matched by treatment
Should I get tested?
What will I do if it’s positive?
What will we do if he’s positive?
Testing is not always matched by treatment:Confidentiality
Health InsuranceEmployers
Family
Where do we go now?
• Try to resolve ethical, legal and social issues– Experts in science, ethics, law.– Policy makers
• Develop new, cheap, simple, effective and accurate tests– Testing for all
• Advance research in disease treatment– Gene therapy– Drug treatment