overview of molecular diagnostics part 1 - laboratory · pdf fileoverview of molecular...

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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





•Sequencing


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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


Quiz

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•PCR enabled•Amplification for quantity

•Target specific enrichment


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•Template DNA

•Primers

•Taq enzyme

•Nucleotide building blocks


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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





•Sequencing


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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





•Sequencing


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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





•Sequencing


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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


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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


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





•Sequencing


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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

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Gene Specific Issues

• RET-PTC and EML4-ALK• Intrachromosomal rearrangements

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Summary





•Sequencing


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