Where is DNA
Whole blood - nuclei of leukocytes (wbcs) - no nuclei in erythrocytes (rbcs)
Proteins + RNA found
Plasma (soluble RNA) - NO genomic DNA
DNA extraction
Plasma and RBCs removed to enrich for wbcs - lysis of wbcs release nucleus = DNA access.
RNA + protein extraction
Also abundant wbcs = extraction simultaneously.
Essential for extraction method
Proper handling = no degradation or contamination
Tri-reagent (TRIzol)
Contains phenol and guanidine isothiocyanate = lyse cells, denature proteins, inactivate RNases
TRIzol with Chloroform (bromochloropropane)
When combined - separate into three phases:
Aqueous (RNA)
Interphase (DNA)
Organic (proteins + lipids)
Guanidine isothiocyanate and phenol
Guanidine salts = RNA protection from degradation
Phenol = dissolves proteins
Aims of Extraction
3 aims to maintain experimental reliability:
1. High conc - sufficient yield for downstream application like PCR/sequencing
2. High purity - min contamination from proteins, salts, phenols
3. Integrity - intact, non-degraded nucleic acids
Quantitation
Quantitation = measure amount + purity of DNA (spectrophotometry - based on UV absorbance e.g. nanodrop)
(both needed for extraction success)
Qualitation
Qualitation = assess DNA integrity and size - agarose/acrylamide gel electrophoresis to visualise band patterns (both needed for extraction success)
NanoDrop Spectrophotometry
Measure UV absorbance (1-2uL of undiluted sample)
Nucleic acids = 260nm
Proteins = 280nm
Beer-Lambert Law to calculate concentration
Rapid analysis (<5 secs), no cuvette, min sample consumption
Gel electrophoresis principle
Separates DNA based on size and charge.
DNA = (-) charge
Migrates from cathode to anode under electric field
Smaller fragments move faster through gel matrix
DNA visualised after stain (e.g. ethidium bromide) under UV light
PCR
Polymerase Chain Reaction
In vitro technique - amplifies specific DNA sequences
- template DNA
- forward + reverse primers
- Taq (DNA polymerase)
- dNTPs
- MgCl2
- Buffer
PCR Cycle
- Denaturation (94°C) - separates DNA strands
- Annealing (50-60°C) - primers bind to target sequences
- Extension (72°C) - Taq polymerase synthesises new strand
30-40 cycles = exponential amplification
RT-PCR
Converts RNA into cDNA using reverse transcriptase before amplification and used for gene expression analysis
qPCR
(Real-Time PCR)
Monitors amplification in real time using fluorescence and provides quantitative data via CT values - required in Prader-Willi testing to detect expression differences accurately
CFTR ∆F508 mutation PCR
3bp deletion in exon 10 of CFTR gene. PCR amplifies region:
Wild type allele - 97bp
Mutant allele - 94bp
10% acrylamide gel required to resolved small size diff
Heterozygotes show both bands
SNRPN and Prader-Willi Syndrome
SNRPN located on chr 15q11-q13 and paternally expressed.
Loss of paternal expression = Prader-Willi Syndrome.
RT-PCR detects SNRPN mRNA - absence/reduction of band = loss of expression rather than methylation status
WASP control use
WASP not imprinted and consistently expressed in hematopoietic cells.
Serves as INTERNAL POSITIVE CONTROL = confirms RNA integrity, cDNA synthesis, PCR success
WASP present + SNRPN absent = true biological absence
Both absent = technical failure
Common Problems (extraction/PCR)
- Low yield = incomplete lysis/poor elution
- low A260/A280 = protein contamination
- low A260/A230 = salt/phenol contamination
- smearing on gel = degraded DNA/excess template
- No bands = enzyme failure/incorrect cycling conditions
