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Overview of Molecular DiagnosticsPart 1
Jennifer L. Hunt, MD, MEdAubrey J. Hough Jr, MD, Endowed Professor of PathologyChair of Pathology and Laboratory MedicineUniversity of Arkansas for Medical [email protected]
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Agenda
•Basic Techniques in Molecular Diagnostics•Difficult starting materials
•Polymerase chain reaction
•Reverse transcriptase – PCR
•Detection methods
•Sequencing
•Fluorescent in situ hybridization
Kary Mullis (1983): Polymerase Chain Reaction (Nobel Prize, 1993)
Watson & Crick (1953): DNA (Nobel Prize, 1962)
Alfred Knudson (1971): Tumor suppressor genes
Frederick Sanger (1975): Sequencing
First human genome sequenced (2000‐2003)
1950
1970
1960 1980
1990
2000
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Source of DNA
• Blood
• Body fluids
• Cytology samples
• Fresh or frozen cells and tissue
• Paraffin embedded tissue• Cell blocks
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Potential Issues With Samples
• Sample purity • Normal cell contamination
• Sample size and template concentration
• Template quality and degradation
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Nucleic Acids in Tissues
What have you done to your specimen?
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Fixatives Are Our Friend
• Stabilize cell morphology and tissue architecture
• Disable proteolytic enzymes
• Strengthen samples to withstand further processing and staining
• Protect samples against microbial contamination and decomposition
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Fixatives in AP
•Cross-linking fixatives•Formaldehyde
•Glutaraldehyde
•Paraformaldehyde
•Precipitating fixatives•Alcohol
•Methanol
•Acetone
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DNA Damage in Formalin
300 to 400Basepair Fragments
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Tissue Fixation
Fixative Components DNA Quality RNA Quality
Unbuffered formalin Formaldehyde Poor Poor
Buffered formalin FormaldehydePhosphate buffers
Fair Fair
Ethanol 70% - 100% Good Good
Decalcifying acids Various acids Poor Poor
Bouin’s Picric acidFormaldehydeGlacial acetic acid
Poor Poor
Mercury solutions Mercuric chloride Sodium acetate
Poor Poor
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DNA Degradation
69%
17%
5%0%
10%
20%
30%
40%
50%
60%
70%
152 b 268 bp 676 bp
Gillio-Tos, Pathology, 39:345, 2007
25 year-old tissue blocks
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Agenda
•Basic Techniques in Molecular Diagnostics•Difficult starting materials
•Polymerase chain reaction
•Reverse transcriptase – PCR
•Detection methods
•Sequencing
•Fluorescent in situ hybridization
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Analysis of DNA
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Analysis of DNA
•Fresh tissue, blood, or cytology fluids
•Frozen tissue
•Fixed tissue
•Paraffin embedded tissue•Archival
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Polymerase Chain Reaction
•Discovered in 1983 (Kary Mullis, Cetus)
•Patented technique
•Patent sold to Roche in 1991 for $300 Million
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•Problems before PCR•Quantity of DNA was small
•The target was a tiny fraction of all DNA
Polymerase Chain Reaction
Quiz
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•PCR enabled•Amplification for quantity
•Target specific enrichment
Polymerase Chain Reaction
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•Template DNA
•Primers
•Taq enzyme
•Nucleotide building blocks
Polymerase Chain Reaction
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Primer Design
•Primers•Short DNA sequence (18-24 nucleotides)
• Forward and Reverse•“Sense & Antisense”
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Primer Design
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Primer Binding
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Polymerase
•Taq Enzyme•Thermus Aquaticus
•DNA polymerase
•Heat activated
TaqTaqTaqTaqTaqTaqTaqTaq
C G AT
C
G
G
G
T
TT
A
A
A
A
CC CTTT AA GG C
G
G
G
T
T
A
A
A
C CGG AAT A G T T T C C CA A G T
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PCR: Thermal Cyclers
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Cycle 2
DenaturationCycle 1
DenaturationAnnealingExtension
90
72
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DenaturationAnnealingExtension Denaturation
A
G
C
T A
G
T
AG
C
T
A
G
C
T
A
GC
T
AG
C
T
AG
C
TAA
G
T
G
C T
A
G
C
T
Annealing
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PCR Amplification
Time
PCR Product Exponential phase
Plateau phase
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Agenda
•Basic Techniques in Molecular Diagnostics•Difficult starting materials
•Polymerase chain reaction
•Detection methods
•Reverse transcriptase – PCR
•Sequencing
•Fluorescent in situ hybridization
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PCR Product Analysis
• What do we do after PCR?• Detect the sequence in the product
• The actual sequence (Sequencing)
• Differences that cause altered migration (MSI)
• Detect the amount of product• Quantitative (real time-PCR)
• Semi-quantitative (LOH)
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PCR product detection
•Gel: Agarose or polyacrylamide
•Capillary electrophoresis
•Quantitative PCR
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Agarose Gel Electrophoresis
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Capillary Electrophoresis
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Capillary Electrophoresis
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Capillary Gel Electrophoresis
2000
1800
1600
1400
Size of PCR product (basepairs)aaaaaaaaaa
aaaaaaaa
Relative amount of PCR product
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Quantitative Real-Time PCR(not RT-PCR)
• Polymerase chain reaction with a reporter• Double stranded DNA binding dye
• Fluorescent reporter probe
Quantitative PCR
Fluorescence
Cycle Number
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Quantitative PCR
Fluorescence
Cycle Number
Fluorescent Reporter Probe
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Agenda
•Basic Techniques in Molecular Diagnostics•Difficult starting materials
•Polymerase chain reaction
•Reverse transcriptase – PCR
•Detection methods
•Sequencing
•Fluorescent in situ hybridization
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Nucleotide
Ribose
Nucleo
side
H
CH2
H
HH
H
O
3’
5’
O
NH2
Cytosine
OH
RNA: Bases (A, C, G, U)
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Gene Structure and Translation
mRNA
DNA
Protein
Intron 1 Intron 2 Intron 3
EX
ON
2
EX
ON
3
EX
ON
4
EX
ON
1
5’ UTRPromoter
3’ UTR
EX
ON
4
EX
ON
3
EX
ON
2
EX
ON
1
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Reverse Transcriptase – PCR
Extract RNAfrom sample
Reverse TranscribemRNA to cDNA
Perform PCR on
cDNA sample
Analyze PCR
products
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Primers for Reverse Transcriptase
• Random hexamers• Anneals randomly
• Oligo dT • Anneals to polyA tail of mRNA
• Product specific primers• Anneals to location of interest
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mRNA
Primers for Reverse Transcriptase
AAAAAA
TTTTTT
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RT
Reverse Transcriptase - PCR
CC C CGGG TT TTT AAAAA GGC CGG AAU A G U U U C C CA A G U
cDNA
mRNA
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RT-PCR Product Analysis
• What do we do after RT-PCR?• Detect qualitative product
• Abnormal transcripts (Translocation analysis)
• Detect the amount of product, which represents the amount of expression of a gene• Quantitative (Real time or quantitative RT-PCR)
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Detecting Translocations
• DNA based PCR testing
• RNA based RT-PCR testing
• Fluorescent in situ hybridization (FISH)
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Gene 1DNA
Gene 2DNA
Fusion mRNA
X
Product
X
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Agenda
•Basic Techniques in Molecular Diagnostics•Difficult starting materials
•Polymerase chain reaction
•Reverse transcriptase – PCR
•Detection methods
•Sequencing
•Fluorescent in situ hybridization
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Mutation Detection Techniques
• PCR detection and screening methods• Heteroduplex formation
• Allele specific PCR
• SSCP
• Full sequencing methods• Sanger sequencing
• Single base extension
• Pyrosequencing
• Next generation sequencing
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Detecting Point Mutations
• Full Gene Sequencing Approaches• Dideoxy sequencing (“first generation”)
• Sanger sequencing
• Single base extension sequencing (“SNaPshot”)
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Sanger Sequencing: The Gold Standard
Template DNA
PCR Product
Sequence
PCR with primers
Sequencing reaction
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Frederick Sanger (1918 - )
• University of Cambridge, England
• Nobel Laureate twice• Insulin and unique protein structures (1958)
• Dideoxy method of sequencing “Sanger method” (1980)
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Sequencing Reaction
• Primer
• Polymerase
• Labeled bases (A T C G)• Abundant normal nucleotides
• Low concentration modified bases • Dideoxy instead of deoxy: stops extension (chain
terminator)
• Added fluorescent tag: product visualization
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Nucleotide
Deoxyribose
Nucleo
side
H
CH2
H
H
HH
H
5’
O
Cytosine
Dideoxynucleotide Chain Terminator
NH2
H
P
P
P
P
P
Dideoxynucleotide Nucleotides
C G ATT
A
CC CTTT AA GG
G
G
G
T
T
A
A
A
C
Sanger Sequencing
C CGG AAT A G T T T C C CA A G T
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Sanger Sequencingttgatttgagattaatctacttttgatttgagattaatctactttgatttgagattaatctacttgatttgagattaatctattgatttgagattaatctttgatttgagattaatcttgatttgagattaatttgatttgagattaattgatttgagattattgatttgagattttgatttgagatttgatttgagattgatttgagttgatttgattgatttgttgatttttgattttgatttgattgttt
A G C T
T T G A T T T G A G A T T A A T C T A C T T
ttgatTttgaTttgA
CC CTTT AA GG
SNaPshot Sequencing
G
T
C
A
C CGG AAT A G T T T C C CA A G T
Patient’s tumorNormal Control
PIK3CA Gene
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Pitfalls in Sequencing
• Performance characteristics• Sensitivity is variable
• Deletion detection not optimal
• Technical• Expensive and labor intensive
• Time consuming
• Tissue fixation may be an issue
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Normal and Mutant
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3/8
2/8
Normal Contamination
100%
75%
50%
Mt Wt Mt Wt Mt Wt Mt Wt
Mt Wt Mt Wt Mt Wt Wt
Mt Wt Mt Wt
Wt
WtWtWtWt
4/8
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Allele Specific PCR
CC CTTT AA GGC CGG AAT A G T T T C C CA A G T
CC CTCT AA GGC CGG GAT A G T T T C C CA A G T
Normal Sequence (A allele)
Mutant Sequence (G allele)
Allele Specific PCR
Allele Specific PCR for BRAF Mutation
Normal BRAF
Mutant BRAF
BRAF gene mutation
Pyrosequencing for BRAF Mutation
Normal BRAF
Mutant BRAF
BRAF gene mutation
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Agenda
•Basic Techniques in Molecular Diagnostics•Difficult starting materials
•Polymerase chain reaction
•Reverse transcriptase – PCR
•Detection methods
•Sequencing
•Fluorescent in situ hybridization
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Basic Techniques
• Fluorescent in situ hybridization• Quantitative
• Amplification
• Deletion
• Qualitative • Translocation analysis
• Presence foreign nucleic acid (virus)
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In Situ: Normal Cell
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In Situ for Amplification
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In Situ for Deletion
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In Situ for Virus
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In Situ for Translocations
• Fusion probes• One probe on each
partner
• Both genes must be known
• Will only pick up consistent partner genes
• Break-apart probes• Probes flank the break
point on one partner
• Only one gene must be known
• Will pick up variable translocations
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Fusion for Translocation
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Break-Apart for Translocation
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Ewing’s Sarcoma, EWS break-apart probe92
Pitfalls in Translocation Detection
• Tissue specific issues• Small size
• Fixation
• Interpretation issues• Truncation
• Overlap
• Gene and probe specific issues