DNA Analysis

1
DNA Analysis

• Dr Tony Fryer
• Department of Clinical Biochemistry
• Centre for Cell and Molecular Medicine
• North Staffordshire Hospital NHS Trust
• University of Keele

2
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?

3
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...

4
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

5
2. Basic Principles of DNA analysis
6
DNA structure

• Double-stranded with 'sense' strand running in
  the opposite direction to the 'antisense' strand.
• Strands connected by hydrogen bonding between
  bases
• AT (2 bonds)
• CG (3 bonds)
• Total number of bases in human sequence 2.3 x
  109
• Approx 50,000 genes.

7
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

8
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.

9
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 (GCAT ratio)

10
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.

11
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'

12
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

13
Southern blotting (b)

• Mutation detection
• Mutation causes loss/gain of restriction site
• Fragment sizes altered
• Different banding patterns observed (RFLP)

14
Southern blotting (c)

• Disadvantages
• Labour intensive
• Expensive
• Use of radioactivity
• Not amenable to automation
• Not suitable for widespread clinical use

15
Polymerase chain reaction (a)

• Denaturation
• Annealing of primers
• Amplification
• Repeat 25 cycles
• 106 copies of a target sequence

ssDNA
16
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
17
Cyclin D1 polymorphism
origin
159bp
139bp
CT CT CT CC TT CC TT CT CC CC
markers
Genotype
18
Polymerase chain reaction (b)

• Advantages
• Uses v. small quantities of DNA
• Relatively cheap
• No requirement for autoradiography
• More amenable to automation
• Widespread clinical applications

19
Polymerase Chain Reaction

• The start of a explosion in interest in DNA
  technology-
• Single gene disorders are the tip of the
  iceberg..

20
Polymerase Chain Reaction

• .but what lies beneath the surface?
• What does the future hold?

21
PCR the future

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

22
3. New developments in technology
23
PCR - possibilities for automation

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

24
PCR Automation - DNA Extraction

• Options Capital cost Cost/sample Throughput
• 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?

25
PCR Automation - Thermal cycling

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

26
PCR Automation - Detection

• Options
• DigestGel 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

27
Amplification Refractory Mutation System (ARMS) -
principle
28
GSTM1 ARMS Assay
1
2
3
4
5
6
7
8
Exon
5
3
132 bp
273 bp
C/G substitution
273 bp
132 bp
110 bp
GSTM1 A
GSTM1 B
GSTM1 AB
GSTM1 null
29
GSTM1 ARMS gel
30
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

31
Automated gel-free detection systems

• Temperature gradient separation
• DASH
• WAVE
• Sequencing
• Pyrosequencing

32
Dynamic Allele Specific Hybridisation

• PCR
• Product immobilization
• Single strand isolation
• Probe hybridisation
• Read fluorescence while heating
• Temperature-dependent melting
• Analysis allele scoring

33
Temperature modulated heteroduplex analysis (WAVE)

• Useful for
• screening for
• unknown
• mutations
• E.g. tumour
• analysis
• More sensitive
• /automated
• than SSCP

34
Fragment separation by WAVE
35
The principle of pyrosequencing (a)
36
The principle of pyrosequencing (b)
37
4. Clinical applications
38
Classical Applications

• Single Gene Disorders such as
• Cystic Fibrosis
• Alpha-1-Antitrypsin Deficiency
• Haemochromatosis
• Molecular diagnostics also applicable to
• Tissue typing
• Viral infection

39
Cystic Fibrosis - background

• 'Single most common autosomal recessive disorder
  among Caucasians.'
• 12500 live births
• Defective Gene
• - Cystic Fibrosis Transmembrane Conductance
  Regulator (CFTR)
• - Chloride Ion Channel
• - Chromosome 7
• - 250,000 base pairs
• - 27 exons
• - 1480 amino acids

40
CF delta-F508 by site-directed mutagenesis of
PCR primers
Homozygous positive
Homozygous negative
Heterozygous carrier
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
41
? Some CF gels in here?
42
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

43
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)

44
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

45
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

46
Can we predict which patients will develop skin
cancer within 5 years?

• Will this affect patient management follow-up?

47
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

48
1.00
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)
49
Genetic factors

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

50
UV
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
51
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 (p0.003, OR11.5)

52
Tumour latency Gene-Environment interactions
53
Targeted surveillance 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

54
Predictive index (PI) - Australian model

• PI (K1.23)(A0.085)(S1.47)(M0.62)-(G1.15)
  -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 CI7.7-31.9), plt0.0001.

55
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

56
5. Where is DNA analysis going in the clinical
laboratory?
57
Clinical molecular genetics - 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

58
Molecular genetics - the future

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

59
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