Association of Molecular Pathology - az9194.vo.msecnd.netaz9194.vo.msecnd.net/pdfs/130401/2.pdf ·...

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)

http://www.reproductive-revolution.com/refs/index.html

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) Nucleic Acids in Tissues

8

What have you done to your specimen?

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)

Disclosure(s) DNA Damage in Formalin

300 to 400

Basepair

Fragments

DNA Damage in Formalin

http://www.internetphotos.net/Single-human-chromosome.html

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







• Sequencing


• Microarrays

16

Disclosure(s) Analysis of DNA

17

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


• Problems before PCR

• Quantity of DNA was small

• The target was a tiny fraction of all DNA

20


• PCR enabled

• Amplification for quantity

• Target specific enrichment

23


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

Disclosure(s) Primer Design

26

Disclosure(s) Primer Binding

27

Disclosure(s) Polymerase

• Taq Enzyme

• Thermus Aquaticus

• DNA polymerase

• Heat activated

28

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

http://upload.wikimedia.org/wikipedia/commons/4/48/Prototype_PCR_thermal_cycler.jpg

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) PCR Amplification

32

Time

PCR

Product Exponential phase

Plateau phase







• Sequencing


• 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)

36

Agarose Gel Electrophoresis

Disclosure(s) Capillary Electrophoresis

37

Disclosure(s) Capillary Electrophoresis

Disclosure(s) Capillary Gel Electrophoresis

39

2000

1800

1600

1400

Size of PCR

product

(basepairs) aaaaaaaaaa

aa

aa

aa

aa

Relative

amount of

PCR product

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) Quantitative PCR

43

Flu

ore

scence

Cycle Number

Disclosure(s) Quantitative PCR

44

Flu

ore

scence

Cycle Number

Disclosure(s) Fluorescent Reporter Probe

45







• Sequencing


• 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) Primers for Reverse Transcriptase

51

mRNA AAAAAA

TTTTTT

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

55

Gene 1

DNA

Gene 2

DNA

Fusion

mRNA

X

Product

X







• Sequencing


• 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

P

P

P

P

P

Dideoxynucleotide

Nucleotides

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

C

C

C

T

T

T

A

A

G

G

SNaPshot Sequencing

G

T C

A

C

C

G

G

A

A

T

A

G

T

T

T

C

C

C

A

A

G

T

Patient’s tumor Normal Control

PIK3CA Gene

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 and Mutant

74

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

Disclosure(s) Allele Specific PCR

76

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) Allele Specific PCR for BRAF Mutation

78

Normal BRAF

Mutant BRAF

BRAF gene mutation

Disclosure(s) Pyrosequencing for BRAF Mutation

79

Normal BRAF

Mutant BRAF

BRAF gene mutation







• Sequencing


• Microarrays

80

Disclosure(s) Basic Techniques


– Quantitative

• Amplification

• Deletion

– Qualitative

• Translocation analysis

• Presence foreign nucleic acid (virus)

81

84

In Situ: Normal Cell




Disclosure(s)

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)

Ewing’s Sarcoma, EWS break-apart probe

Disclosure(s) Pitfalls in Translocation Detection

• Tissue specific issues

• Small size

• Fixation

• Interpretation issues

• Truncation

• Overlap

• Gene and probe specific issues

93

Disclosure(s) Gene Specific Issues

• RET-PTC and EML4-ALK

• Intrachromosomal rearrangements

98

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

Disclosure(s) Arrays

103

Feature

(probe)

Target

(sample)

Label

Surface

substrate

Test DNA

Labeled with Cy3

Control DNA

Labeled with Cy5

Hybridize

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

Ratio

Cy3/Cy5

Ratio analysis

Disclosure(s) Summary






• Sequencing


• Microarrays

118

Association for Molecular Pathology 9650 Rockville Pike

Bethesda, MD 20814

[email protected]

www.amp.org

© Association for Molecular Pathology, 2013 119

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