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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. 1. Background 2. Principles of DNA analysis - Basic principles - Techniques

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DNA Analysis

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  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. 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.

  7. 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

  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 (GC:AT 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. 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

  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 Genotype CT CT CT CC TT CC TT CT CC CC markers

  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 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?

  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 • 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

  27. normal mutant Normal DNA Normal DNA common common No amplifiction PCR product No PCR product Amplification Refractory Mutation System (ARMS) - principle

  28. 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

  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 Disorderssuch 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.' • 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

  40. 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

  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. 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 Clinical risk factors

  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

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