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DNA Analysis. Dr Tony Fryer Department of Clinical Biochemistry & Centre for Cell and Molecular Medicine North Staffordshire Hospital NHS Trust & University of Keele. Overview. 1. Background 2. Principles of DNA analysis - Basic principles - Techniques

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Dna analysis

DNA Analysis

Dr Tony Fryer

Department of Clinical Biochemistry

& Centre for Cell and Molecular Medicine

North Staffordshire Hospital NHS Trust

& University of Keele


Overview
Overview

1. Background

2. Principles of DNA analysis

- Basic principles

- Techniques

3. New developments in technology

4. Novel applications - from single gene disorders to high risk patient identification

5.Where is DNA analysis going in the clinical laboratory?


1 background the current role of dna based tests
1. BackgroundThe current role of DNA-based tests

Generally used for:-

  • single gene disorders

  • small populations (rare diseases individually)

  • patient diagnosis

    But this restricted applicability is changing…...


Genetics revolution
Genetics revolution

  • Increased public awareness

  • Improvements in technology

  • Greater understanding of genetic basis of disease

    • Human genome project

  • Increased interest from clinicians

  • More requests for genetic tests



Dna structure

5’

3’

3’

5’

DNA structure

  • Double-stranded with 'sense' strand running in the opposite direction to the 'antisense' strand.

  • Strands connected by hydrogen bonding between bases:

    A:T (2 bonds)

    C:G (3 bonds)

  • Total number of bases in human sequence = 2.3 x 109

  • Approx 50,000 genes.


Gene structure

5’

3’

Gene structure

  • Exon - encodes mRNA.

  • Intron - between exons.

    - spliced out during mRNA production.

  • Promoter - TAATA or Goldberg-Hogness Box.

    - binding site for RNA polymerase.

    - site of action of some hormone/receptors.

  • CAT Box - upstream control element (CCAAT Box).

    - essential for accurate initiation of transcription.

  • Enhancers - 5', 3' or intragenic.

    - Regulate level of expression of genes.

  • CAP site - Transcription initiation point.

    - caps mRNA - stabilises & ensures accurate translation.

  • Poly A site - applies poly A tail to mRNA (stability & transport).

    Mutation at any of these points can result in aberrant protein synthesis


The effect of mutation
The Effect of Mutation

Normal base sequence:-

The man had one son and his dog was red but his son had one sad cat.

Substitution:-

The man had one son and his dog was red but his son hid one sad cat.

Deletion:-

The man had one son and hsd ogw asr edb uth iss onh ado nes adc at.

Insertion:-

The man had one son and his dog was red bus yth iss onh ado nes adc at.

Nonsense:-

The man had one son end.

Splice site mutations:-

The man had one wqt oen uts jfi pwx jei jsd pke zso nan dhi sdo gwa sre dbu thi sso nha don esa dca t.

Trinucleotide repeats:-

The man had one son and his dog was red but but but but but but but but but but his son had one sad cat.


Hybridisation a
Hybridisation (a)

  • Concept central to the understanding of molecular biology.

  • Relates to the hydrogen bonding between strands of DNA.

  • Antisense strand = complementary to the sense strand:

    5'-CCGGTCATTGCCAAGGT-3'

    3'-GGCCAGTAACGGTTCCA-5'

  • The two strands can be split (denatured) by heat and re-anneal (hybridise) spontaneously when the temperature drops below the melting temperature (Tm)

    Tm depends on:-

    1. Length of DNA sequence

    2. Composition (GC:AT ratio)


Hybridisation b
Hybridisation (b)

  • Under some circumstances (low stringency), non-identical DNA sequences may hybridise:-

    1. At lower temperatures

    2. At high salt concentrations

  • stringency determines specificity.


Restriction enzymes
Restriction enzymes

  • Naturally-occurring enzymes which cut DNA at specific sequences (often palindromic)

    Examples:

  • EcoRI (Sticky ends)

    5'-GAATTC-3' 5'-G + AATTC-3'

    3'-CTTAAG-5' 3'-CTTAA G-5'

  • SmaI (Blunt ends)

    5'-CCCGGG-3' 5'-CCC + GGG-3'

    3'-GGGCCC-5' 3'-GGG CCC-5'

    MboI 5'-GATC-3'

    MstII 5'-CCTNAGG-3'


Southern blotting a
Southern blotting (a)

  • Digestion of DNA with restriction enzyme

  • Separation of fragments by gel electrophoresis

  • Transfer to a nylon/nitrocellulaose membrane

  • Detection of sequence of interest by a radio-labeled probe

  • Autoradiography


Southern blotting b
Southern blotting (b)

Mutation detection

  • Mutation causes loss/gain of restriction site

  • Fragment sizes altered

  • Different banding patterns observed (RFLP)


Southern blotting c
Southern blotting (c)

Disadvantages

  • Labour intensive

  • Expensive

  • Use of radioactivity

  • Not amenable to automation

  • Not suitable for widespread clinical use


Polymerase chain reaction a

No of copies

No of cycles

Polymerase chain reaction (a)

ssDNA

  • Denaturation

  • Annealing of primers

  • Amplification

  • Repeat 25 cycles

  • 106 copies of a target sequence


Cyclin D1 gene

Exons:

1

2

3

4

5

3’

5’

C

T

1722

159 bp

PCR product

Hae

III restriction site

20 bp

139 bp

Banding patterns following

Hae

III restriction

CC

CT

TT

159 bp

139 bp


Cyclin d1 polymorphism
Cyclin D1 polymorphism

origin

159bp

139bp

Genotype

CT CT CT CC TT CC TT CT CC CC markers


Polymerase chain reaction b
Polymerase chain reaction (b)

Advantages

  • Uses v. small quantities of DNA

  • Relatively cheap

  • No requirement for autoradiography

  • More amenable to automation

  • Widespread clinical applications


Polymerase chain reaction

Polymerase Chain Reaction

The start of a explosion in interest in DNA technology:-

Single gene disorders are the tip of the iceberg…..


Polymerase chain reaction1

Polymerase Chain Reaction

….but what lies beneath the surface?

What does the future hold?


Pcr the future
PCR: the future

  • Opening the door to new technology

  • Opening the door to new applications



Pcr possibilities for automation
PCR - possibilities for automation

Stages in DNA analysis by PCR:

  • DNA extraction

  • Thermal cycling

  • Product detection


Pcr automation dna extraction
PCR Automation - DNA Extraction

Options: Capital costCost/sampleThroughput

Phenol/Chloroform low £0.30 10 samples/h

Alkaline low £0.15 20 samples/h

Extraction kit

(e.g. Nucleon) low £2 20 samples/h

Automated system high ?£2 100 samples/h

……but is extraction necessary?


Pcr automation thermal cycling
PCR Automation - Thermal cycling

Scaling down

  • 0.5ml tubes

  • 0.2ml tubes

  • 96/384 well plates

  • Capillaries (Light cycler)

    Robotics


Pcr automation detection
PCR Automation - Detection

Options

  • Digest+Gel electrophoresis

  • ARMS

  • DASH – allele specific labeled probes

  • Pyrosequencing – mini sequence analysis

  • WAVE (Temperature Modulated Heteroduplex Analysis)

  • Real-time PCR (e.g. Light cycler)

  • Mass Specrometry

  • Chip technology


Amplification refractory mutation system arms principle

normal

mutant

Normal DNA

Normal DNA

common

common

No amplifiction

PCR product

No PCR product

Amplification Refractory Mutation System (ARMS) - principle


GSTM1 ARMS Assay

Exon

1

2

3

4

5

6

7

8

5’

3’

273 bp

132 bp

C/G substitution

273 bp

132 bp

110 bp

GSTM1 A

GSTM1 B

GSTM1 AB

GSTM1 null



Amplification refractory mutation system arms advantages
Amplification Refractory Mutation System (ARMS) - advantages

  • No requirement for restriction digestion

  • Opportunities for multiplex analysis

    • E.g. Elucigene CF20 kit

      But…..

  • Requires more Taq polymerase

  • Still dependent on gel separation of PCR products


Automated gel free detection systems
Automated gel-free detection systems

  • Temperature gradient separation

    • DASH

    • WAVE

  • Sequencing

    • Pyrosequencing


Dynamic allele specific hybridisation
Dynamic Allele Specific Hybridisation

  • PCR

  • Product immobilization

  • Single strand isolation

  • Probe hybridisation

  • Read fluorescence while heating

  • Temperature-dependent melting

  • Analysis & allele scoring


Temperature modulated heteroduplex analysis wave
Temperature modulated heteroduplex analysis (WAVE)

  • Useful for

  • screening for

  • unknown

  • mutations

  • E.g. tumour

  • analysis

  • More sensitive

  • /automated

  • than SSCP






Classical applications
Classical Applications

Single Gene Disorderssuch as:

  • Cystic Fibrosis

  • Alpha-1-Antitrypsin Deficiency

  • Haemochromatosis

    Molecular diagnostics also applicable to:

  • Tissue typing

  • Viral infection


Cystic fibrosis background
Cystic Fibrosis - background

  • 'Single most common autosomal recessive disorder among Caucasians.'

  • 1:2500 live births

  • Defective Gene:

    - Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

    - Chloride Ion Channel

    - Chromosome 7

    - 250,000 base pairs

    - 27 exons

    - 1480 amino acids


Cf delta f508 by site directed mutagenesis of pcr primers
CF: delta-F508 by site-directed mutagenesis of PCR primers

Heterozygous

carrier

Homozygous

positive

Homozygous

negative

Heteroduplex fragments

217bp

202bp

The delta-F508 mutation results in the loss of a

phenyalanine residue at amino acid 508

and accounts for around 80% of CF chromosomes



Cystic fibrosis the classical single gene disorder
Cystic Fibrosis - the classical single gene disorder?

  • Over 500 mutations in the CFTR now identified

  • Mutation frequency depends on ethnic origin

  • Demonstrates significant variation in phenotype:

    Phenotype-Genotype Correlation

    Genotype% Pancreatic Insufficiency

    F508/F508 99

    F508/Other 72

    Other/Other 36

  • But even with the same causative mutation, phenotype differs dramatically

  • Do genetic factors predispose to severe disease even within single gene disorders? - Modifier genes


Future applications
Future Applications

  • Pharmacogenetics

  • Tumour analysis - oncogenes, TSG

  • Detection of rearrangements - e.g. Philadelphia chromosome

  • Detection of residual disease

  • Strain typing

  • Chromosomal aberrations - FISH

  • SNP analysis

    • genetic predisposition to disease

    • disease severity/prognosis (even in single gene disorders)


Renal transplant recipients a growing population
Renal transplant recipients - a growing population

  • World-wide increase in functioning transplants

    • improved patient management - longer graft survival

    • inproved access to transplantation

  • Number of UK renal allograft recipients:

    • 11,700 in 1994

    • 18,400 in 1999

  • Growing population who will develop complications of long term immunosupression


Non melanoma skin cancer a major complication
Non-melanoma skin cancer - a major complication

  • Increased incidence

    • 20-fold for basal cell carcinoma (BCC)

    • 200-fold for squamous cell carcinoma (SCC)

  • More aggressive behaviour

    • Present earlier

    • more numerous

    • grow more rapidly

    • metastisise earlier

  • 5% of recipients will die as a consequence of these maligancies


Can we predict which patients will develop skin cancer within 5 years

Can we predict which patients will develop skin cancer within 5 years?

Will this affect patient management & follow-up?


Clinical risk factors

UV within 5 years?

Latitude

Outdoor occupation

Sunbathing habits

Cumulative sun exposure

Holidays abroad

Gender

Skin type 1

Blue or green eyes

Red/blonde hair color

Immunosuppression

Degree

Regimen

Duration

Other

Smoking (SCC)

Premalignant lesions

Arsenic exposure

Clinical risk factors


1.00 within 5 years?

AK negative

0.75

Proportion tumor-free

0.50

AK positive

0.25

0.00

0

10

20

30

Time from transplantation to appearance of first NMSC (years)


Genetic factors
Genetic factors within 5 years?

  • UV-induced oxidative stress

  • Melanisation

  • Immune modulation

  • Detoxification of smoking-derived chemicals

  • Cell-cycle control


UV within 5 years?

Mn-SOD

EC-SOD

ROS

Immunomodulation

Melanisation

TNF-

IL-10

TGF-

IFN-

Tyr

Lipid and DNA

hydroperoxides

CYP2D6

MC1R

VDR

GSTM1

GSTT1

GSTM3

GSTP1

Smoking

Cell cycle

control

Cyclin D1


Gene environment interactions
Gene-environment interactions within 5 years?

What effect does exposure have on associations of GSTM1 null with skin cancer risk?

  • GSTM1 null effect most evident in those with:

    • High UV exposure (p=0.003, OR=11.5)


1.00 within 5 years?

0.75

Other genotype/sunbathing

score combinations

Proportion tumor free

0.50

GSTM1 null+sunbathing score>3

0.25

0.00

0

5

10

15

20

Time post transplantation (years)

Tumour latency: Gene-Environment interactions


Targeted surveillance the predictive index
Targeted surveillance: within 5 years?The predictive index

  • Use stepwise logistic regression to obtain the best set of predictors for developing NMSC within 5 or 10 years

  • Generate a predictive index (score) that identifies high risk patients


Predictive index pi australian model
Predictive index (PI) - Australian model within 5 years?

PI = (K*1.23)+(A*0.085)+(S*1.47)+(M*0.62)-(G*1.15)-5.88

  • K= Actinic keratoses pre Tx; 1 if any present, 0 if absent

  • A = Age at transplantation

  • S = Skin type; 1 if type 1, 0 if types 2-4

  • M = Gender; 1 if male, 0 if female

  • G = GSTT1 genotype; 1 if null, 0 if A

    If the score is -1.4 or greater, the model predicts a squamous cell tumour within 5 years while if the score is less than -1.4, no tumour is predicted.

  • Accuracy = 78.4%

  • Sensitivity = 82.0% PPV = 46.3%

  • Specificity = 77.5%. NPV = 94.8%

  • odds ratio = 15.7 (95% CI=7.7-31.9), p<0.0001.


  • Predictive index clinical application
    Predictive index within 5 years?- clinical application

    These indices can be simplified and applied to clinical management settings to:

    • identify high risk patients for entry into clinical surveillance programmes

    • target appropriate treatments

    • enable focusing of resources

    • ?amend immunosuppresive dose



    Clinical molecular genetics the future
    Clinical molecular genetics within 5 years?- the future

    • Will include very large numbers of patients

      • every clinical speciality

    • Includes areas other than just diagnosis

      • management

      • monitoring

      • treatment

    • Applicable to patients of every age (not just children)

      Advances in technology will bring DNA analysis to the DGH


    Molecular genetics the future
    Molecular genetics - the future within 5 years?

    Will the new applications provide sufficient workload to warrant establishment of a new Clinical Biochemistry sub-speciality?


    A few final tips

    A few final tips….. within 5 years?

    1. Almost all DNA analyses require an EDTA sample.

    Cytogenetics require heparin.

    If in doubt, request both!

    2. Always ask for a family history and ethnic origin of the patient


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