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DNA Separation Techniques , Screenshot 2023-04-14 at 10.21.13 am,…
DNA Separation Techniques
Separate DNA
PCR creates a mixture of products
Up to 48 different sized products can be created
Electrophoresis - Resolution of 1bp
Electrophoresis
Electrical charges carried by molecules
Phosphate backbone readily gives up H+
Makes DNA negatively charged in most buffers
Two main types
Gel Electrophoresis
Polyacrylamide
Higher resolution
Agarose
General separation
Capillary Electrophoresis
Why use CE for DNA Analysis?
Automated - Injection, separation, detection
Rapid separations
Excellent sensitivity & resolution
Internal size standard
Steps
1. Injection
- Electrokinetic injection
ssDNA - formamide as part of prep
Current generated by voltage & resistance experienced will draw DNA- into capillary
Draws in other neg charged ions
2. Separation
- Through entangled polymer (POP-4 or 6)
Size based separation
Polymer length & concentration determines separation characteristics
3. Detection
- Using argon laser & CCD camera
PCR product fluorescently labelled following amplification
Diff. kits use diff. fluorescent dyes
Detect many diff. dyes
Sizing Methods
Global Southern Method
Generates best-fit curve from
all matched fragments
in the size standard
Local Southern Method
Generates best-fit curve from only
nearby internal lane standard
data points
Uses 4 fragments of internal standard to unknown fragment to generate standard curve
Interpretation
Analysis used to be Genescan Analysis & Genotyper
Genescan - analysed raw data from polyacrylamide gel
Genotyper - took data & analysed it against size standards & allelic ladder
Gene Mapper
incorporates both
Comprises mixture analysis software
Overview
Data collection
Colour Separation
Peak identification
Peak Sizing
Comparison to Allelic Ladder
Genotype Assignment to Alleles
Data Review by Analyst/Examiner
Confirmation of Results by Second Analyst/Examiner
Problems with profiles
1. CE Issues
Spikes
Instrument related
Non-reproducible
Fluctuation in voltage/air in capillary
Narrow
Dye blobs
Unincorporated dye can get into capillary
Characteristic peaks
Chemistry related
Reproducible
Dissociated primer dye
Wide & jagged in appearance
Preferential injection/Degradation
Smaller products are injected more
Enviro exposure degrades DNA via random strand breakage. Lose larger strands first
Over injection
Noise/Random Peaks
Instrument related
Non-reproducible
Background
2. Biological Artefacts
Stutters
Caused by activity of Taq pol
Chemistry related
One repeat unit shorter (backstutter) or longer (upstutter) than parent peak
More stutter - longer repeat units
N and N+1 peaks
Caused by incomplete adenylation
Non-template Addition/Shoulder
Pull-up peaks
Caused by over-amplified PCR products
Out-of-date matrix
Sample Renaturation
Off-Ladder Alleles & Tri-alleles
Threshold Settings
Detection Limit: 3x the stdv of noise
Dynamic range: Range of sample quantities lowest - highest
Stochastic Threshold: Level of quantifiable DNA below which peaks can show severe imbalance
