Genomes

Genomics: study of the whole set of DNA base sequences in the cell of organisms of a particular species

Genes: sections of DNA which code for the production of polypeptides or proteins (1.5% of human DNA is in the form of genes) 

Non-coding (junk) DNA: carry out regulatory functions

Comparing Genomes helps:

• Gives information on the location of genes
• Understand the role of genetic information on health and behaviour
• Provide evidence for the evolutionary links between organisms

1) The genome is broken up and sequenced in sections of about 750 base pairs

2) Sequencing carried out several times on overlapping fragments

3) Overlapping fragments analysed

4) Fragments are put back together to form a complete code

Restrictions Endonucleases - Cut DNA into smaller fragments

Gel Electrophoresis - DNA fragments sorted by size

DNA Probes - Specific sequences on DNA fragments located

DNA fragments analysis - Specific base sequences identified

Ligase Enzymes - DNA fragments annealed

They recognise specific recognition sequences that are palindromic and cut/digest though each sugar phosphate backbone of DNA at this point - Different recstriction enzymes have different active sites which are complementary to different sequences (EcoRI cuts at GAATTC whereas HindIII cuts at AAGCTT)

Recognition sequences are at either side of the DNA fragments - DNA is incubated with a specific restriction enzyme which cut the DNA fragment by hydrolysis. The fragment may have sticky ends (a short section of unpaired bases) which can bind or anneal the fragment to another piece of DNA with a complementary sequence

used to separate DNA fragments by size according to base length (accurate to 1 base)

1)Gel Plate of Agarose Sugar
2)Electrodes attached to each end of the plate
3)Wells are cut into the negative end of the plate
4)DNA Samples are put into the wells
5)Gel is immersed in a tank of buffer solution and a current is passed through for 2 hours
6) DNA is attracted to the anode as the phosphate groups in DNA are negatively charged
7) The shortest lengths of DNA move further than longer fragments in the alloted time

Viewing Fragments

• The DNA is initially labelled with fluorescent markers so that after electrophoresis, the bands can be viewed under UV light
• Southern Blotting - Nylon (nitrocellulose) sheet placed over the gel, covered in paper towels, pressed and left overnight. DNA fragments transferred to the sheets are made visible if the DNA is labelled with radioactive markers, and photographic film is then placed over the sheet

...binds to a single strand of complementary DNA by annealing...

Probes are used in:

• finding genes for genetic modification
• identifying the same gene in different species when comparing genomes
• looking for alleles for a disease

Radioactive Probes:

• used to bind to specific ssequences of DNA is order to identify their location
• 50-80 nucleotides long - includes a phosphate groups which shows them up on photographic film
• The radioactive probe binds to the specific complementary bases and are then seen on photographic film

Fluorescent Markers - probes show up when UV light is shone on the fragment

A surface with different DNA probes fixed to it

Reference and test DNA (knwon and unknown sequences) are labelled with fluorescent markers and applied to the microarray. Complementary DNA is annealed to the fixed probes. The microarray is treated and scanned by a computer to see if both smaples have a particular DNA sequence

Sequencing and Copying DNA - polymerase chain reactions are used to make multiple copies (amplified) of DNA fragments. Only used on short fragments - it needs primers and must use heating adn cooling. it is useful in forensic work (hair and blood but not RBCs)

Primers - short pieces of single stranded DNA, complementary to bases at the start of the DNA fragment to copy

It relies on: antiparallel backbones  in DNA strands, strands having a 5' end and 3'end, growing from the 3' end, AT and CG base pairing

Method

1) Double stranded DNA fragments, free nucleotides, primers and DNA polymerase are heated to 95dc to break hydrogen bonds
2) Mix cooled to 55dc to allow primers to anneal to DNA strands
3) Mixture heated to 72dc to allow DNA Polymerase to line up the free nucleotides to complementary base pairs on the strand
4) Two new complementary strands of DNa are formed

• Process is repeated and DNA amounts increase exponentially

1) Extract DNA
2)Restriction enzymes cut the DNA (750bp max length)
3) PCR
4) Chain Termination Method: Primers, DNA polymerase, free nucleotides and modified nucletides (ddNTP) - a hydroxyl group is removed so they can not form hydrogen bonds end the chain stops where these nucleotides bond with the strand of DNA 
5)Tubes are added to an electrophoresis plate to separate the strands
6) Read from the bottom up to find the sequence - it is repeated 6-10times before the sequence is deemed accurate

Automated Sequencing

Fragments of varying lengths are produced with fluorescent markers as the last added base - each nucleotide is tagged with a different colour

The sequence is shown by the order of colours as electrophoresis separates the fragments by length

The fragments are different lengths because 1 out of every 100 free nucletides is modified (ddNTP or didNTP) to stop the DNA strand from growing

1) Gemone cut into small fragments using restriction enzymes

2) Fragments are inserted into bacterial artificial chromosomes BACs

3) BACs with different fragments in each are inserted into bacteria so each bacterium contains a different BAC

4) The bacteria divide, creating colonies of cloned cells that all contain a specific DNA fragment. THe different colonies make a complete genome library

5) DNA is extracted from each colony and cut using restriciton enzymes, producing overlapping piecies of DNA

6) Pieces are sequenced, using the CTM, and the pieces are put back together to giver the fulll sequence from the BAC

7) All the fragmetns from all the BACs are put back in order, by computers, to complete the genome