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Disclosure(s)
The Association for Molecular Pathology Education. Innovation and Improved Patient Care. Advocacy.
www.amp.org
Molecular Pathology Review Course Basic Techniques Overview
Jennifer L. Hunt, MD, MEd Aubrey J. Hough Jr, MD, Endowed Professor of Pathology
Chair of Pathology and Laboratory Medicine University of Arkansas for Medical Sciences
April 2013
Disclosure(s) Disclosure(s)
In accordance with ACCME guidelines, any individual in a position to influence and/or control the content of this ASCP CME activity has disclosed all relevant financial relationships within the past 12 months with commercial interests that provide products and/or services related to the content of this CME activity.
The individual below has responded that he/she has no relevant financial relationship(s) with commercial interest(s) to disclose:
Jennifer L. Hunt, MD, MEd
2
Disclosure(s) Agenda
• Basic Techniques in Molecular Diagnostics
• Difficult starting materials
• Polymerase chain reaction
• Reverse transcriptase – PCR
• Detection methods
• Sequencing
• Fluorescent in situ hybridization
• Microarrays
3
Disclosure(s) 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
Disclosure(s) Source of DNA
• Blood
• Body fluids
• Cytology samples
• Fresh or frozen cells and tissue
• Paraffin embedded tissue
• Cell blocks
6
Disclosure(s) Potential Issues With Samples
• Sample purity
• Normal cell contamination
• Sample size and template concentration
• Template quality and degradation
7
Disclosure(s) Fixatives Are Our Friend
9
• Stabilize cell morphology and tissue architecture
• Disable proteolytic enzymes
• Strengthen samples to withstand further processing and staining
• Protect samples against microbial contamination and decomposition
Disclosure(s) Fixatives in AP
10
• Cross-linking fixatives
• Formaldehyde
• Glutaraldehyde
• Paraformaldehyde
• Precipitating fixatives
• Alcohol
• Methanol
• Acetone
Disclosure(s) DNA Damage in Formalin
300 to 400
Basepair
Fragments
DNA Damage in Formalin
Disclosure(s) Tissue Fixation
14
Fixative Components DNA Quality RNA Quality
Unbuffered formalin Formaldehyde Poor Poor
Buffered formalin Formaldehyde
Phosphate buffers
Fair Fair
Ethanol 70% - 100% Good Good
Decalcifying acids Various acids Poor Poor
Bouin’s Picric acid
Formaldehyde
Glacial acetic acid
Poor Poor
Mercury solutions Mercuric chloride
Sodium acetate
Poor Poor
Disclosure(s) DNA Degradation
15
• Text
69%
17%
5%0%
10%
20%
30%
40%
50%
60%
70%
152 b 268 bp 676 bp
Gillio-Tos, Pathology, 39:345, 2007
Disclosure(s) Agenda
• Basic Techniques in Molecular Diagnostics
• Difficult starting materials
• Polymerase chain reaction
• Reverse transcriptase – PCR
• Detection methods
• Sequencing
• Fluorescent in situ hybridization
• Microarrays
16
Disclosure(s) Analysis of DNA
• Fresh tissue, blood, or cytology fluids
• Frozen tissue
• Fixed tissue
• Paraffin embedded tissue
• Archival
18
Disclosure(s) Polymerase Chain Reaction
19
• Discovered in 1983 (Kary Mullis, Cetus)
• Patented technique
• Patent sold to Roche in 1991 for $300 Million
Disclosure(s) Polymerase Chain Reaction
• Problems before PCR
• Quantity of DNA was small
• The target was a tiny fraction of all DNA
20
Disclosure(s) Polymerase Chain Reaction
• PCR enabled
• Amplification for quantity
• Target specific enrichment
23
Disclosure(s) Polymerase Chain Reaction
24
• Template DNA
• Primers
• Taq enzyme
• Nucleotide building blocks
Disclosure(s) Primer Design
• Primers
• Short DNA sequence (18-24 nucleotides)
• Forward and Reverse
•“Sense & Antisense”
25
Taq Taq Taq Taq Taq Taq Taq Taq
C G
A
T
C
G
G
G
T
T
T
A
A
A
A
C
C
C
T
T
T
A
A
G
G
C
G
G
G
T
T
A
A
A
C
C
G
G
A
A
T
A
G
T
T
T
C
C
C
A
A
G
T
Disclosure(s) PCR: Thermal Cyclers
30
Disclosure(s)
Cycle 2
Denaturation Cycle 1
Denaturation Annealing Extension
90
72
55
Denaturation Annealing Extension Denaturation
A
G
C
T A
G
T
A G
C
T
A
G
C
T
A
G C
T
A G
C
T
A G
C
T A A
G
T
G
C T
A
G
C
T
Annealing
Disclosure(s) Agenda
• Basic Techniques in Molecular Diagnostics
• Difficult starting materials
• Polymerase chain reaction
• Reverse transcriptase – PCR
• Detection methods
• Sequencing
• Fluorescent in situ hybridization
• Microarrays
33
Disclosure(s) 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)
34
Disclosure(s) PCR product detection
• Gel: Agarose or polyacrylamide
• Capillary electrophoresis
• Quantitative PCR
35
Disclosure(s) Quantitative Real-Time PCR (not RT-PCR)
42
• Polymerase chain reaction with a reporter
• Double stranded DNA binding dye
• Fluorescent reporter probe
Disclosure(s) Agenda
• Basic Techniques in Molecular Diagnostics
• Difficult starting materials
• Polymerase chain reaction
• Reverse transcriptase – PCR
• Detection methods
• Sequencing
• Fluorescent in situ hybridization
• Microarrays
46
Disclosure(s) RNA: Bases (A, C, G, U)
47
Nucleotide
Rib
ose
N
ucle
osid
e
H
CH2
H
H H
H
O
3’
5’
O
NH2
Cytosine
OH
Disclosure(s) 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’ UTR
Promoter
3’ UTR
EX
ON
4
EX
ON
3
EX
ON
2
EX
ON
1
Disclosure(s) Reverse Transcriptase – PCR
49
Extract RNA
from sample
Reverse
Transcribe
mRNA
to cDNA
Perform
PCR on
cDNA sample
Analyze
PCR
products
Disclosure(s) Primers for Reverse Transcriptase
50
• Random hexamers
• Anneals randomly
• Oligo dT
• Anneals to polyA tail of mRNA
• Product specific primers
• Anneals to location of interest
Disclosure(s) Reverse Transcriptase - PCR
52
RT
C
C
C
C
G
G
G
T
T
T
T
T
A
A
A
A
A
G
G
C
C
G
G
A
A
U
A
G
U
U
U
C
C
C
A
A
G
U
cDNA
mRNA
Disclosure(s) RT-PCR Product Analysis
53
• 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)
Disclosure(s) Detecting Translocations
• DNA based PCR testing
• RNA based RT-PCR testing
• Fluorescent in situ hybridization (FISH)
54
Disclosure(s) Agenda
• Basic Techniques in Molecular Diagnostics
• Difficult starting materials
• Polymerase chain reaction
• Reverse transcriptase – PCR
• Detection methods
• Sequencing
• Fluorescent in situ hybridization
• Microarrays
56
Disclosure(s) 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
57
Disclosure(s) Detecting Point Mutations
• Full Gene Sequencing Approaches
• Dideoxy sequencing (“first generation”)
• Sanger sequencing
• Single base extension sequencing (“SNaPshot”)
58
Disclosure(s) Sanger Sequencing: The Gold Standard
59
Template DNA
PCR Product
Sequence
PCR with primers
Sequencing reaction
Disclosure(s) Frederick Sanger (1918 - )
• University of Cambridge, England
• Nobel Laureate twice
– Insulin and unique protein structures (1958)
– Dideoxy method of sequencing “Sanger method” (1980)
60
Disclosure(s) 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
61
Disclosure(s) Dideoxynucleotide Chain Terminator
62
Nucleotide
Deo
xy
rib
ose
Nu
cleosid
e
H
CH2
H
H
H H
H
5’
O
Cytosine
NH2
H
C G
A
T T
A
C
C
C
T
T
T
A
A
G
G
G
G
G
T
T
A
A
A
C
Sanger Sequencing
C
C
G
G
A
A
T
A
G
T
T
T
C
C
C
A
A
G
T
Sanger Sequencing ttgatttgagattaatctactt
ttgatttgagattaatctact
ttgatttgagattaatctac
ttgatttgagattaatcta
ttgatttgagattaatct
ttgatttgagattaatc
ttgatttgagattaat
ttgatttgagattaa
ttgatttgagatta
ttgatttgagatt
ttgatttgagat
ttgatttgaga
ttgatttgag
ttgatttga
ttgatttg
ttgattt
ttgatt
ttgat
ttga
ttg
tt
t
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
ttgatT
ttgaT
ttgA
Disclosure(s) 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
73
Disclosure(s) Normal Contamination
75
3/8
2/8
75%
50%
Mt Wt Mt Wt Mt Wt Wt
Mt Wt Mt Wt
Wt
Wt Wt Wt Wt
100% Mt Wt Mt Wt Mt Wt Mt Wt 4/8
C
C
C
T
T
T
A
A
G
G
C
C
G
G
A
A
T
A
G
T
T
T
C
C
C
A
A
G
T
C
C
C
T
C
T
A
A
G
G
C
C
G
G
G
A
T
A
G
T
T
T
C
C
C
A
A
G
T
Normal Sequence (A allele)
Mutant Sequence (G allele)
Allele Specific PCR
Disclosure(s) Agenda
• Basic Techniques in Molecular Diagnostics
• Difficult starting materials
• Polymerase chain reaction
• Reverse transcriptase – PCR
• Detection methods
• Sequencing
• Fluorescent in situ hybridization
• Microarrays
80
Disclosure(s) Basic Techniques
• Fluorescent in situ hybridization
– Quantitative
• Amplification
• Deletion
– Qualitative
• Translocation analysis
• Presence foreign nucleic acid (virus)
81
84
In Situ: Normal Cell
86
In Situ for Amplification In Situ for Amplification
87
In Situ for Deletion
88
In Situ for Virus
Disclosure(s) In Situ for Translocations
89
• 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
Disclosure(s) Fusion for Translocation
Disclosure(s) Break-Apart for Translocation
Disclosure(s) Pitfalls in Translocation Detection
• Tissue specific issues
• Small size
• Fixation
• Interpretation issues
• Truncation
• Overlap
• Gene and probe specific issues
93
100
Agenda
•Basic Techniques in Molecular Diagnostics
•Difficult starting materials
•Polymerase chain reaction
•Reverse transcriptase – PCR
•Detection methods
•Sequencing
•Fluorescent in situ hybridization
•Microarrays
100
Microarray
Tissue
Microarray
What is on the array?
Oligo-
nucleotide
What is measured?
Expression
Microarray
Gene copy
numbersSNPs
TissueNucleic
Acids
Disclosure(s) Different Types of Arrays
• Targets for hybridization
• Oligonucleotides (25 – 85 basepairs)
• BAC (bacterial artificial chromosomes, 80-200 basepairs)
• cDNA
• Types of arrays
• Whole genome
• Targeted
102
Jennifer Hunt, MD | 114 [email protected]
HARVARD MEDICAL SCHOOL MASSACHUSETTS GENERAL HOSPITAL HARVARD MEDICAL SCHOOL MASSACHUSETTS GENERAL HOSPITAL
Disclosure(s) Interpretation and Analysis
• Ratio of fluorescence calculated and overall value given for each spot
• Heat maps with 3 data points are generated
• Gene target
• Sample identity
• Value of fluorescence (continuous scale)
115
Disclosure(s) Summary
• Basic Techniques in Molecular Diagnostics
• Difficult starting materials
• Polymerase chain reaction
• Reverse transcriptase – PCR
• Detection methods
• Sequencing
• Fluorescent in situ hybridization
• Microarrays
118
Association for Molecular Pathology 9650 Rockville Pike
Bethesda, MD 20814
www.amp.org
© Association for Molecular Pathology, 2013 119