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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Dr Tony Fryer
Department of Clinical Biochemistry
& Centre for Cell and Molecular Medicine
North Staffordshire Hospital NHS Trust
& University of Keele
2. Principles of DNA analysis
- Basic principles
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?
Generally used for:-
But this restricted applicability is changing…...
A:T (2 bonds)
C:G (3 bonds)
- spliced out during mRNA production.
- binding site for RNA polymerase.
- site of action of some hormone/receptors.
- essential for accurate initiation of transcription.
- Regulate level of expression of genes.
- caps mRNA - stabilises & ensures accurate translation.
Mutation at any of these points can result in aberrant protein synthesis
Normal base sequence:-
The man had one son and his dog was red but his son had one sad cat.
The man had one son and his dog was red but his son hid one sad cat.
The man had one son and hsd ogw asr edb uth iss onh ado nes adc at.
The man had one son and his dog was red bus yth iss onh ado nes adc at.
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.
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.
Tm depends on:-
1. Length of DNA sequence
2. Composition (GC:AT ratio)
1. At lower temperatures
2. At high salt concentrations
5'-GAATTC-3' 5'-G + AATTC-3'
3'-CTTAAG-5' 3'-CTTAA G-5'
5'-CCCGGG-3' 5'-CCC + GGG-3'
3'-GGGCCC-5' 3'-GGG CCC-5'
No of cyclesPolymerase chain reaction (a)
III restriction site
Banding patterns following
CT CT CT CC TT CC TT CT CC CC markers
The start of a explosion in interest in DNA technology:-
Single gene disorders are the tip of the iceberg…..
….but what lies beneath the surface?
What does the future hold?
Stages in DNA analysis by PCR:
Options: Capital costCost/sampleThroughput
Phenol/Chloroform low £0.30 10 samples/h
Alkaline low £0.15 20 samples/h
(e.g. Nucleon) low £2 20 samples/h
Automated system high ?£2 100 samples/h
……but is extraction necessary?
No PCR productAmplification Refractory Mutation System (ARMS) - principle
Single Gene Disorderssuch as:
Molecular diagnostics also applicable to:
- Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
- Chloride Ion Channel
- Chromosome 7
- 250,000 base pairs
- 27 exons
- 1480 amino acids
The delta-F508 mutation results in the loss of a
phenyalanine residue at amino acid 508
and accounts for around 80% of CF chromosomes
Genotype% Pancreatic Insufficiency
Will this affect patient management & follow-up?
UV within 5 years?
Cumulative sun exposure
Skin type 1
Blue or green eyes
Red/blonde hair color
Arsenic exposureClinical risk factors
1.00 within 5 years?
Time from transplantation to appearance of first NMSC (years)
UV within 5 years?
Lipid and DNA
What effect does exposure have on associations of GSTM1 null with skin cancer risk?
1.00 within 5 years?
Proportion tumor free
GSTM1 null+sunbathing score>3
Time post transplantation (years)
Tumour latency: Gene-Environment interactions
PI = (K*1.23)+(A*0.085)+(S*1.47)+(M*0.62)-(G*1.15)-5.88
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.
These indices can be simplified and applied to clinical management settings to:
Advances in technology will bring DNA analysis to the DGH
Will the new applications provide sufficient workload to warrant establishment of a new Clinical Biochemistry sub-speciality?
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