DNA AnalysisDNA Analysis

Dr Tony FryerDr Tony FryerDepartment of Clinical Biochemistry

& Centre for Cell and Molecular Medicine

North Staffordshire Hospital NHS Trust & University of Keele

OverviewOverview1.1. BackgroundBackground2.2. Principles of DNA analysisPrinciples of DNA analysis

- Basic principles- Techniques

3.3. New developments in technologyNew developments in technology4.4. Novel applications - from single gene Novel applications - from single gene

disorders to high risk patient identificationdisorders to high risk patient identification5.Where is DNA analysis going in the clinical 5.Where is DNA analysis going in the clinical

laboratory?laboratory?

1. Background1. 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 revolutionGenetics 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 testsMore requests for genetic tests

2.2. Basic Principles of DNA analysisBasic Principles of DNA analysis

• 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.

DNA structureDNA structure

5’3’

3’5’

Gene structureGene 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

5’ 3’

The Effect of MutationThe Effect of MutationNormal 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 sequence2. Composition (GC:AT ratio)

Hybridisation (b)Hybridisation (b)

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

1. At lower temperatures2. At high salt concentrations

• stringency determines specificitystringency determines specificity.

Restriction enzymesRestriction 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)Polymerase chain reaction (a)

• Denaturation

• Annealing of primers

• Amplification

• Repeat 25 cycles

• 106 copies of a target

sequence

ssDNA

No of cyclesN

o of

cop

ies

CC CT TT

Cyclin D1 gene

139 bp

159 bp

Hae III restriction site

5’ 3’

Banding patterns following Hae III restriction

159 bp

PCR product

20 bp 139 bp

Exons: 1 2 3 4 5

C1722T

Cyclin D1 polymorphismCyclin D1 polymorphism

159bp139bp

CT CT CT CC TT CC TT CT CC CC markers

origin

Genotype

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

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

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

Polymerase Chain Reaction

….but what lies beneath the surface?

What does the future hold?

PCR:PCR: the future the future

• Opening the door to new technology • Opening the door to new applications

3.3. New developments in New developments in technologytechnology

PCR - possibilities for automationPCR - possibilities for automation

Stages in DNA analysis by PCR:• DNA extraction• Thermal cycling• Product detection

PCR Automation - DNA Extraction

Options: Capital cost Cost/sample Throughput

Phenol/Chloroform low £0.30 10 samples/hAlkaline low £0.15 20 samples/hExtraction kit (e.g. Nucleon) low £2 20 samples/hAutomated system high ?£2 100 samples/h

……but is extraction necessary?

PCR Automation - Thermal cycling

Scaling down• 0.5ml tubes• 0.2ml tubes• 96/384 well plates• Capillaries (Light cycler)

Robotics

PCR Automation - DetectionOptions• 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 Amplification Refractory Mutation System (ARMS) - principle(ARMS) - principle

common

mutant

Nor

mal

DN

A

No amplifiction

No PCR product

common

normal

Nor

mal

DN

A

PCR product

5’ 3’

1 2 3 4 5 6 7 8

C/G substitution

GSTM1 ARMS Assay

132 bp

Exon

273 bp

GSTM1 A GSTM1 B GSTM1 AB GSTM1 null110 bp

273 bp

132 bp

GSTM1 ARMS gelGSTM1 ARMS gel

Amplification Refractory Mutation System Amplification Refractory Mutation System (ARMS) - advantages(ARMS) - advantages

• No requirement for restriction digestion• Opportunities for multiplex analysis

– E.g. Elucigene CF20 kit

But…..But…..• Requires more Taq polymerase• Still dependent on gel separation of PCR products

Automated gel-free detection systemsAutomated gel-free detection systems

• Temperature gradient separation– DASH– WAVE

• Sequencing– Pyrosequencing

Dynamic Allele Specific HybridisationDynamic 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 Temperature modulated heteroduplex analysis (WAVE)(WAVE)

•Useful for screening for unknown mutations

•E.g. tumour analysis

•More sensitive/automated than SSCP

Fragment separation by WAVEFragment separation by WAVE

The principle of pyrosequencing (a)The principle of pyrosequencing (a)

The principle of pyrosequencing (b)The principle of pyrosequencing (b)

4.4. Clinical applicationsClinical applications

Classical ApplicationsClassical Applications

Single Gene DisordersSingle Gene Disorders such as:such as:– Cystic Fibrosis– Alpha-1-Antitrypsin Deficiency– Haemochromatosis

Molecular diagnostics also applicable to:Molecular diagnostics also applicable to:– Tissue typing– Viral infection

Cystic Fibrosis - backgroundCystic Fibrosis - background• 'Single most common autosomal recessive disorder 'Single most common autosomal recessive disorder

among Caucasians.'among Caucasians.'• 1:2500 live births1:2500 live births• Defective Gene: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 CF: delta-F508 by site-directed mutagenesis of PCR primersmutagenesis of PCR primers

Homozygouspositive

202bp217bp

Homozygousnegative

Heterozygouscarrier

Heteroduplex fragments

The delta-F508 mutation results in the loss of a The delta-F508 mutation results in the loss of a phenyalanine residue at amino acid 508 phenyalanine residue at amino acid 508 and accounts for around 80% of CF chromosomesand accounts for around 80% of CF chromosomes

? Some CF gels in here?

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

• Over 500 mutations in the CFTR now identified• Mutation frequency depends on ethnic origin• Demonstrates significant variation in phenotype:Phenotype-Genotype CorrelationGenotype % Pancreatic InsufficiencyF508/F508 99F508/Other 72Other/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• 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 disorderseven in single gene disorders)

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

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

Will this affect patient management & follow-up?

Clinical risk factors

• UV – 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

Prop

ortio

n tu

mor

-free

Time from transplantation to appearance of first NMSC (years)0 10 20 30

0.00

0.25

0.50

0.75

1.00

AK negative

AK positive

Genetic factors

• UV-induced oxidative stress• Melanisation• Immune modulation• Detoxification of smoking-derived chemicals• Cell-cycle control

UV

ROS

Lipid and DNAhydroperoxides

GSTM1GSTT1GSTM3GSTP1

Immunomodulation

TNF-IL-10TGF-IFN-

Melanisation

MC1RVDR

Mn-SODEC-SOD

CYP2D6

SmokingCyclin D1 Cell cyclecontrol

Tyr

Gene-environment interactions

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)

Prop

ortio

n tu

mor

free

Time post transplantation (years)0 5 10 15 20

0.00

0.25

0.50

0.75

1.00

Other genotype/sunbathing score combinations

GSTM1 null+sunbathing score>3

Tumour latency: Gene-Environment interactions

Targeted surveillance: The predictive indexThe 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 modelPredictive index (PI) - Australian modelPI = (K*1.23)+(A*0.085)+(S*1.47)+(M*0.62)-(G*1.15)-5.88PI = (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

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

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

Clinical molecular genetics Clinical molecular genetics - the future- 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 DGHAdvances in technology will bring DNA analysis to the DGH

Molecular genetics - the future

Will the new applications provide sufficient workload to warrant establishment of a new

Clinical Biochemistry sub-speciality?

A few final tips…..

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