01 Methods of genetic testing

Use of DNA extraction

Molecular technique to look at changes in the DNA sequence

Why is blood used for DNA extraction?

1. Easy to obtain

2. Body replaces it very easily

3. Enough DNA obtained with small volumes

Limitation of DNA extraction

1. Not all DNA is the same in all parts of the body

2. Inhereted muttations will be seen in blood, but acquired ones, unless they are blood related, you will not see in the mutation

Process of DNA extraction

1. Lysis of RBC using NH4Cl

2. Washing to remove Hb

3. Lysis of WBC using strong chaotropic agents e.g. SDS, high salt concentration, phenol

4. DNA in aqueous phase is precipitated by isopropanol

5. DNA washing in 70% ethanol

6. Air-dry DNA pellet and resuspend

Quality of DNA is determined by

Integrity

Purity

Electrophoresis definition

The movement of a charged molecule, within a fluid or gel, under the influence of an electric field Used to separate particles based on charge and size

DNA is separated by eelctrophoresis based on ___

Size

All DNA is negatively charged

Electrophoresis of DNA- process

DNA (and RNA) is negatively charged, moves towards the positive electrode.

The distance moved by a DNA molecule depends on the size of the fragment.

Smaller fragments will migrate faster

Agarose

A polysaccharide obtained from agar

Resolution of agarose

Low

Polyacrylamide

A polymer of acrylamide; chemical reaction

Resolution of acrylamide

High

Use of agarose gel

DNA Separation

Use of polyacrylamide gel

Used for DNA or protein separation

Movement of DNA in agarose is:

• Horizontal

• Quick

• Poor resolution

How is agarose gel prepared?

1. Mix agarose and buffer

2. Microwave to boi

3. Cool to 65deg and pour into mold

   1. Comb to make wells

Loading dyes are important because

They colour the solution

They contain sucrose to make them heavy so the DNA will sink into the bottom of the well

They move under the influence of the electric field giving an indication of the distance moved

Sucrose in loading dyes

Increases the viscosity of samples, so DNA sample doesn't mix with buffer

Loading gels move under the influence of the electric field

Independently from DNA

DNA stops moving through the gel if

The electrif field is switched off

Gels are visualised by

Staining with a DNA binding chemical such as Ethidium bromidee

Ethidium bromide

Sticks itself to the double strand

Fluoresces when exposed to UV light

Polyacrylamide gels used to

Check site of DNA not integrity

Polyacrylamide

A polymer of acrylamide and bis-acrylamide Has to be poured vertically because it needs the support

Movement of DNA in polyacrylamide:

• Vertical

• Slow

• Very good resolution

Polyacrylamide gels and resolution

Give more resolution than agarose gel as you can see base pair differences in allele samples

Capillary electrophoresis

Same principle as gel electrophoresis but in a narrow bore capillary

Sample volume in capillary eelctrophoresis

10 ul

Two methods for DNA quantification?

• Spectrophotometry

• Fluorescence

Advantage and disadvantage of spectrophotometry

Adv: reads a whole spectrum of wavelengths thus can show protein and solvent contamination

Dis: any nucleic acid will absorb at 160nm; so you don't know if its DNA or RNA for example

Advantage and disadvantage of Fluorescence

Adv: you have DNA to RNA specific dyes

Dis: you get no information of protein or solvent contamination

What are restriction endonucleases?

Enzymes which cut DNA at specific sequences

What do restriction endonucleases do?

1. Restrict or limit viral bacterial infections

2. Cut in the middle of a DNA fragment

3. Cut nucleic acids (DNA)

Restriction enzymes synthesised by bacteria

Destroy foreign DNA such as that of an attacking phage

Methyl groups at the restriction sites

Block the restriction enzyme and protect the bacterial DNA from being cleaved

What makes restriction enzymes a very powerful tool?

That there are hundreds of different enzymes which recognise different DNA sequence and precisely cut them

Methods including uses of restriction enzymes?

1. Southern blotting

2. Genotyping by restriction fragment length polymorphism (RFLP)

3. Cloning

Western blotting

The identification of differences in protein lengths, quantities or modifications. Used in research.

PCR

Exponential amplification of short DNA sequences in vitro

What is needed for PCR?

1. Template DNA

2. Primers

3. DNTPs (dATP, dTTP, dCTP, dGTP)

4. DNA polymerase

5. PCR buffer, ions esp. MgCl2, and water

How are primers designed?

Should be:

• 18-20nt in length (20nt has a good chance of beign a unique sequence in the genome)

• Similar melting temperature (T_M)

• Specific and unique

• No cross or self homologies (should not bind to themselves)

• No stretches of the same nucleotides (slippage)

• Between 45% and 55% GC content

• Contain no known SNPs

The melting temperature is

The point at which half of the DNA molecules have actually melted

Single strand DNA absorbs

More than double stranded DNA

What does self homology in primers lead to?

Hairpin structure

Why should primers be unique?

If not unique, a PCR product will form and will act as a primer to itself

Primers should not include

SNPs

What can mismatches lead to?

Primer annealing

Selective preferential amplification

A suitable primer site must have

• Balanced AT and GC content

• No SNPs [there are mutations]

• No long tracts of the same or similar nucleotides [e.g. polyprimidine tract of only TS and Gc]

Steps of PCR

1. Denaturation 5mins at 95°C

2. Annealing for 1 min at 50-60°C (T_M)

3. Extension/ Elondation for 1min at 72°C

Equation for melting temperature

T_M= 2(A+T) + 4(G+C)

PCR is done on a

Thermal cycler