Week 2- DNA Analysis

What are the three main stages of DNA preparation for analysis

1. Homogenation/lysis

   • Dependent on whether uni or multicellular organism

   • Breaking down tissue in order to access DNA

   • Get a homogenate/a slurry

2. Purification

   • Removal of debris

   • To get pure DNA

   • Treatment to remove lipids (chloroform)

   • Precipitate removes proteins

   • DNAases break down DNA

   • Detergents to remove membranes

   • Needs organic solvent e.g. phenol

3. Precipitation/isolation

What are the ways of analysing

Electropheresis or absorbance

Why is quality control and quantification important during analysis

If the DNA is not good enough the gene sequences will not work

Why are we able to calculate DNA concentrations using absorbance

Nitrogen containing bases have alternating double and single bonds (Like benzene)

= delocalisation of electrons above and below plane that can absorb energy of light

What is the average wavelength that a base absorbs

260nm

What does short wavelength mean

Higher energy

What does the beer-lambert law mean for nucleic acids

Can convert absorbance at 260nm to concentrations

What is the beer-lambert law

A= ε*c*l

• A= absorbance

  ε= molar extinction coefficient

  I= light pathlength (how much light through sample)

How can you calculate the concentration of DNA in a dilution

C= A_(260nm) /ε_(260nm)/ I _(cm)

The extinction coefficient can be converted into standard coefficient multipliers for a 1cm pathway

If used in place of extinction coefficient what is the beer Lambert equation for double stranded DNA

C (dsDNA) = A_(260nm) * 50μg/mL

What is one of the main problems when calculating cones using absorbance

There are other wavelengths than 260nm where other molecules absorb

e.g. polysaccharides and phenols (from prep must be washed) absorb at 230

proteins absorb at 280

How can we check accuracy

The absorbance at 260/280 should be about 1.8

The absorbance at 260/230 should be about 1.5

What is electrophoresis

Movement of charged molecules in an electric field

Way to visualise DNA effectively

Why are we able to use electrophoresis to analyse DNA

Phosphate sugar backbone has a negative charge therefore entire DNA molecule has negative charge

What are the stages of electrophoresis

1. Gel preparation

   • 0.5-2% agarose dissolved in buffer- Provides solid framework for molecule to move

   • Add ethidium bromide to a concentration of 0.5μg/ml- binds to DNA to make it visible

2. Cast the gel

   • Heat and mix to dissolve agarose

   • Cool and poor into casting tray with comb to form loading wells

3. Run the gel

• Add loading dye to DNA samples

• Insert gel tray into apparatus

• Fill container with running buffer

• Load DNA/dye samples into wells

• Load marker

• Start the run

What is the direction of DNA movement along the gel

DNA is negatively charged so will move to positive

How can you visualise agarose gel under UV light

Measure bands using pre-existing scale under scale using calibrator

What happens to larger fragments of DNA

Shorter migration

What influences DNA migration

1. Size of DNA molecules

2. Agarose concentration

3. DNA confromation (other DNA e.g. plasmids arent linear but circular so resist movement)

4. Voltage applied

5. Presence of ethidium bromide/type of argarose/ electrophoresis buffer