week 4 DNA extraction

biochemical markers

• Blood groups, HLA, Serum Proteins &Red Cell enzymes ​

• Low polymorphism but still have utility

Molecular/DNA genetic markers

• Currently very popular​

• High polymorphism and Finest level of variation​

• Relatively easy to analyse

DNA polymorphism

• a sequence difference compared to a reference standard that is present in at least 1–2% of a population​

• can be single bases or thousands of bases​

• may or may not have phenotypic effects​

• Repetitive DNA changes nucleotide length bp (size)

tandem repeats

copies which lie adjacent to eachother, either directly or inverted ​

minisatellite

repeat units from about 10 to 60base pairs, found in many places in the genome, including the centromeres

microsatellite

repeat units of less than 10 base pairs; this includes telomeres, which typically have 6 to 8 base pair repeat units​

transposable elements

• Retrotransposons​

• LTR-retrotransposons (HERVs)​

• Non LTR-retrotransposons​

• SINEs (Short Interspersed Nuclear Elements (Alu))​

• LINEs (Long Interspersed Nuclear Elements (LINE-1))

length polymorphisms

• RFLPs, (Restriction Fragment Length Polymorphisms (variable bp))Original method of SNP analysis​

• VNTRs (Variable number of tandem repeats, large size polymorphisms (10-100bp), required high quality DNA, rarely analysed in normal research/practice) ​

• STRs (Short tandem repeats (2-13 bp multiple times))​

• Alu (SINE (50-500bp) and LINE (~ 7000bp) interspersed polymorphisms)​

DNA extraction

To obtain DNA in a relatively purified form which can be used for further investigations

1. Cellular lysis via disruption of cellular membranes​

2. Protein removal (lysate clearing)​

3. DNA isolation from the remaining solution

DNA sources

• Any nucleated cell!​

• Body fluids: Blood, semen, saliva, sweat, urine​

• Tissues:​

• Hair roots​

• Teeth/bones, faeces, dandruff, ear wax​

• Fingerprints

• Amount of DNA varies: Blood (30mg/mL), semen (250mg/mL) , Saliva (5mg/mL), Hair (750ng/per hair)​

DNA quantity

• A diploid cell contains approximately 6 pg of DNA​

• On average an adult has 5 - 10 X 106 WBC per ml of blood. Therefore, the theoretical recovery of DNA per µL of blood is 30 - 60 ng​

• The PCR reactions call for on average 1-50 ng of DNA (single or double stranded)

history of DNA extraction

• First performed by Friedrich Miescher in 1869​-isolated a material rich in phosphorus, from white blood cells, which he termed “nuclein” and later became nucleic acid​

• Meselson and Stahl (1958) established a more refined technique utilising gradient centrifugation​

• Sambrook and Russell popularised the phenol-chloroform organice xtraction method in the 1980’s​

• In 1991, Lahiri and Nurmberger introduced the use of Proteinase K to help digest proteins ​

• Since 2000, there has been a rapid rise in newer methods (silica columns, magnetic beads, etc.)

cellular lysis

• Breaking of the cellular membranes

• If required in a plant cell or certain tissue types the cell can also be disrupted by mechanical force

• Detergents breaks down the cell membranes​

• Detergents disrupt membranes due to the amphipathic (having both hydrophilic and hydrophobic regions) nature of both cellular membranes and detergent molecules. The detergent molecules pull apart the membranes

• the contents of the cells are distributed in solution (cellular lysate)​

lysate clearing

• to remove proteinaceous material​

• commonly achieved by denaturation and precipitation of proteins​

• also precipitate the DNA from our cellular supernatant we can term the process “differential denaturation” or “differential precipitation”​

• First part of precipitation can use phenol/chloroform or specific salts and buffers to remove the proteins from the DNA​

DNA isolation

• DNA isolation involves the addition of salts​

• The salts interrupt the hydrogen bonds between the water and DNA molecules​

• The DNA is then precipitated using isopropanol or ethanol​

• In the presence of cations, ethanol induces a structural change in DNA molecules that causes them to aggregate and precipitate out of solution​

• The DNA is commonly pelleted by spinning with a centrifuge and the supernatant removed

selection of DNA isolation method

• The selection of the most suitable method for sample preparation depends on the type of sample and the purpose of the molecular analysis​

Factors for consideration:​

• Cost​

• Simplicity and experience

• DNA quality / yield​

• Throughput​

• Compatibility (downstream applications)​

Phenol/Chloroform/Isoamyl alcohol (PCI)

• Place the sample in tube​

• Add SDS (lysis) and Proteinase K, Rupture cells & Breakdown proteins (~24 hours, overnight)​

• Add PCI, Separate protein & DNA, PRECIPITATE DNA with Isopropanol/Ethanol​

• Dilute in Water or TE and store for further analysis​

• Yields high molecular weight DNA – suitable for all applications​

• Time consuming (~30 hours), Hazardous chemicals, Many Transfers (errors)

salting out

• Isolate cell nuclei pellets

• Overnight Proteinase K digestion​

• Saturated (chaotropic) salt solution to “salt out” digested protein (DNA highly hydrophilic due to phosphate backbone)​-retain supernatant

• DNA precipitation by mixing supernatant in ice cold absolute ethanol​

• Dilute in Water or TE and store for further analysis​

• High salt = possible contamination ​

Solid Phase Silica/Spin Column ​

• Collect – Epithelial cells from Buccal cavity or other cells (blood)​

• Lyse - Disruption of most cells is done by chaotropic salts,detergents or alkaline denaturation to produce a cellular lysate​

• Bind - DNA contained within the lysate will bind to the silicacolumn in the presence of chaotropic salts and optimum pH​

• Wash – Post binding the resulting lysate is cleared bycentrifugation or filtration​

• Elute – removal of the salt solution to enable the DNA to bereleased by the silica bed

Chelex Method​

• Chelating resin Removes Magnesium (inactivates DNA nucleases)​

• Use with PCR only (highly alkaline solution)​

• Add sample to the tube ​

• Add water & 5-20% Chelex solution and incubate at 56°C​

• 100°C to rupture cells and destroy proteins​

• Supernatant added directly to PCR​

• DNA is denatured into single strands (stability)

FTA cards/FTA elute

• Chemical coatings of FTA cards (proprietary)​

• DNA can remain bound to the card orbe eluted using water and heat​

• Direct amplification (no washing)​

• Standard amplification (with washing; the so-called “punch-in” method—analyzing DNA while it is on the paper)​

• Extraction or purification of DNA from the card​

• Expensive & proprietary

magnetic beads​

• Following cell lysis, magnetic beads are added to the cell lysate.​

• The magnetic beads are designed to have a high affinity to nucleic acids. In the presence of specific salts, DNA will hybridise to the beads.​

• An external magnetic field can then be applied causing the beads (and the bound DNA) to adhere to the sides of the container.​

• This immobilizes the DNA while allowing the unbound contaminants to be washed away.​

• The beads are washed multiple times to remove any remaining impurities or contaminants, ensuring a high purity of the extracted DNA.​

• An elution buffer (low salt) can then be applied which purifies the DNA from the beads.

key considerations

• Not all tissue / cell types will be compatible with the different extraction methods.​

• You need to think about the characteristics of the tissue / sample when selecting what extraction method to use.​

Example accessible tissues:​

• Blood, buccal swab, hair follicle, saliva,skin scrape, faecal / urine sample, nail clippings​