DNA Manipulation

• Chemical nature
  • acidic, water soluble
• DNA extraction
• Lambda max = wavelength at which a solution can absorb maximum light.
• Charged molecule

Ration of absorbance at 260nm and 280nm tells us if there is protein contamination.

Running DNA on agarose gel separates the fragments of different sizes, smaller fragments move down the gel faster than larger ones.

From prokaryotes and viruses:

• Restriction enzymes
• Ligases
• Reverse transcriptase
• DNA repair and replication enzymes
  • DNA polymerase

All 'natural' enzymes with a perfectly normal function in vivo.

Nucleases

• Cut within a DNA sequence
• At a restricted number of sites
  • Recognition sequence
  • Palindrome
• Named according to the organism that they were purified from. 

Genomic DNA + restriction enzyme

• Smear of different sized fragments

Plasmid DNA + restriction enzyme

• Set of distinct fragments
• A plasmid cut with an enzyme that has a single cut site will generate one fragment

Needs...

• **DNA to act as a template for the polymerase
• A primer to allow the DNA polymerase to start
• dNTPs, Mg2+ etc.
• This is the basis of the PCT reaction and DNA sequencing

Chemical nature of DNA

• ds ==> **DNA easily by heat or change to alkaline conditions
• Once **, DNA can hybridise to another **DNA.
• Only complementary base pairings is allowed
• **DNA + **DNA ==> dsDNA

• dsDNA + heat ==> ssDNA
• Add short ssDNA primer
• Primers will anneal where the sequences are complementary
• Add DNA polymerase
• The polymerase will use the ds primer/sample hybrid to start the reaction
• Reaction always travels from 5' to 3'
• Reaction continues until stopped
• Now there are 2 ds molecules
• Heat added, ds ==> ssDNA
• Now there are 4 ssDNA molecules
• Repeat, 2nd cycle - add primers, extend.
• Repeat... the dsDNA fragments of set size will only be seen at the end of the third cycle.
• Multiple copies of a DNA fragment, all of the same length will be generated
• Determined by the distance between where the primers hybridise
• Detect using a simple agarose gel/EthBr/UV

• Generates multiple copies of dsDNA fragments of the same size
• Does not need much DNA
• Does not have to be of high quality
• Need to take care with:
  • Choice of primer
  • Length of the fragment isolated will vary
  • Polymorphisms
  • Primers can be chosen to identify each chromosome

Uses of PCR:

• Identification of individuals
  • Genetic finger printing
  • Genetic profiling
• Elimination of crime suspects
• Paternity testing
• Identification of crime/accident victims
• Population studies

Based on DNA polymerase...

• Various methods, many work by replicating a DNA strand in the presence of labelled dNTPs.
• Use dideoxynucleotides (ddNTPs) in small amounts in parllel reactions.
• These stop the reaction when on is incorporated

4 parallel reactions:

• dNTPs plus one of the following
  • ddATP
  • ddTTP
  • ddCTP
  • ddGTP
• The polymerase will extend until, at random, one of the ddATPs is incorporated, this will stop the reaction. 

• Probe: DNA fragment
  • radioactive: 32P, 3H
  • non-radioactive: biotin, peroxidase, fluorescent dyes
• dsDNA ==> ssDNA
• hybridise to other ssDNA fragments or to chromosomes

FISHing:

• use of labelled probes to specific DNA sequences located only on ONE chromosome
• only that chromosome pair will fluoresce when viewed under the correct lighting conditions.

Southern Blotting

• electrophoresis perfromed, gel is covered with nitrocellulose sheet.
• pattern on gel is copied or 'blotted' onto the nitrocellulose
• blotted nitrocellulose is incubated it radioactively labelled nucleic acid, then rinsed.
• photographic film is laid over the sheet and is exposed only in areas that contain radioactivity. It is examined for radioactive bands which indicate hybridisation of the original nucleic acids.

Starting DNA

• Genomic DNA (library - all DNA sequences)
• cDNA (library - sequences derived from mRNA from that cell)
• subcloning

Plasmids

• selective markers
  • Tetr and Ampr anitbiotic resistance markers
• transformation
• knowledge of bacterial (E.coli) genetics

Bacteriophages & Artificial Chromosomes

• occur naturally but have been modified to give the vectors

Hosts

• E.coli
• Other prokaryotes
• Viruses
• Animal cells
• Funcal cells, yeast
• Plant cells
• Zygotes, early animal embryo, ovum
• Whole animals

• DNA from two sources is isolated and cleaved with the same restriction endonuclease
• The two types of DNA can pair at their sticky ends when mixed together; DNA ligase joins the segments.
• Plasmids are inserted into bacterial cells by transformation; bacterial cells reproduce and form clones
• Clones can then be screened for gene of interest