Applications of DNA technology Human Genome Project Human Therapeutics Forensic Uses Agriculture

History Late 1980’s idea was proposed Predicted it would take 15 years Cost about $200 million per year $1 per base pair Officially began in 1990 26 June 2000 joint announcement from Blair and Clinton ‘the draft complete’ Joint publication in Nature and Science 12 Feb 2001 14 Apr 2003 – The finished human genome Discuss here the links between the public and private sector. Worth noting that the Wellcome Trust through the creation of the Sanger Institute was responsible for sequencing 1/3 of the DNA sequence.

Why? ‘If I were to study the carburettor of my car engine, even in exquisite detail, I would still have no idea about the overall function of the engine, much less the entire car. To understand what an engine is for, and how it works, I’d need to study the whole thing – I’d need to place the carburettor in context, as one function part among many. The same is true of genes…..’ James Watson

Method Genetic Mapping Physical Mapping DNA sequencing Identifies relative positions of genes E.g. Gene 2 lies between genes 1 and 3 Physical Mapping Absolute positions of genes on chromosomes E.g. Gene 2 is 1 million bp from gene 1 DNA sequencing Actual ATCG combinations Mention the understanding that computers and technology would improve over time therefore speed would increase and cost decrease. Also mention the practise gained by sequencing genomes of smaller organisms such as the nematode, yeast and mice. Compare naming all the houses between edinburgh and London in the correct order. Genetic mapping looks at the towns and gets them in the correct order, physical mapping knows exactly how far apart these towns are from one another, then get down to looking at the houses and where they lie in relation to the sequence.

Polymerase Chain Reaction Critical to the HGP was the ability to create large amounts of DNA for sequencing. PCR is a process to amplify DNA DNA is heated to 95oC The DNA is denatured causing the two strands to separate A primer (short length of DNA) binds (or anneals) to the template strands [after the solution is cooled] Complementary DNA strands form [through the action of DNA polymerase] Lead into this by explaining that we will look at the methods in more detail, but in the end it wasn’t the invention of new methods that sped up the sequencing process, but the automation of old processes. Amplification of DNA used to be done by inserting the required DNA sequence into plasmids of bacteria and then culturing the bacteria. PCR was a process invented by Kary Mullis. Early problems with the method included the need to add more polymerase after each cycle, as DNA polymerase is denatured at 95oC. This problem was solved by thermus aquaticus bacteria (taq polymerase)

Discuss the need for primers (DNA polymerase can only act on double stranded molecules). Highlight how primers are sequences created artificially. We can sequence only the gene wanted from a larger chunk of DNA just by choosing the primer correctly

Genetic Mapping Genetic mapping utilises cross over frequencies between known genetic markers. A genetic marker is any sequence of genome that shows difference between individuals. It could be A gene A Microsatellite or Short Tandem Repeat Differences in genes didn’t give enough information – often they were looking for mutations in the gene and therefore looked for families that were carriers and sufferers of genetic diseases. Turned to STRs which are frequent throughout the genome to give markers.

Microsatellites (STRs) Repeating sequences 2 – 4 nucleotide bases found in mostly in introns (‘junk’ DNA) The number of repeats varies from person to person, but follow patterns of Mendalian inheritance These sequences can be identified using probes. A probe is A short, single stranded sequence of DNA Complementary to DNA base sequence ‘Tagged’ – radioactively or using fluorescent dye Radioactive tags show up with exposure to photographic film. Fluorescent dye shows up when exposed to UV light.

Recombinance / Cross Over Frequency What do you remember about linked genes from Higher. What information do recombinant individuals give us about the relative position of genes? Cross over frequency is the number of recombinant individuals / total number of offspring. Recombinants are different from parental types.

Physical Mapping Use of restriction enzymes Restriction enzymes belong to the group ‘endonucleases’ or nucleases. They cut DNA at specific sequences. Chromosomes can therefore be cut into sequences of different lengths. By using combinations of restriction enzymes and working out the size of the fragments, a pattern of recognition sites in the DNA can be pieced together. Note that there are many different types of physical maps at different resolutions – the ultimate being a total DNA sequence. Recall nucleases break phosphodiester bonds (i.e. break down nucleic acids). Endonucleases are isolated from bacteria, in which it is believed they are used in cellular defences against invading viruses.

Gel electrophoresis Uses physical properties of DNA (size and charge) to separate molecules Gel electrophoresis involves running an electric current through an agarose gel. DNA is loaded into wells at the negative end. DNA is repelled, and moves through the agarose gel at different speeds depending on the size of a fragment. A Molecular weight marker (of known fragment size) is also used. Samples can be compared to this marker to work out their size. Note the many different use of gel electrophoresis, but highlight its importance in this case in determining the size of the fragments.

Physical Mapping cont… M = molecular weight marker 1 = undigested sample 2 = digested with NotI 3 = digested with BamHI 4 = digested with NotI and BamHI 5 = something totally different

Physical Mapping cont…

Physical Mapping cont….

DNA Sequencing Sanger method (aka dideoxy chain-termination method) An unknown DNA template strand is replicated using a primer (to initiate replication), DNA polymerase, nucleotide bases and one of 4 dideoxy nucleotide bases (ddA, ddG, ddC and ddT)

DNA sequencing cont… If a ddNTP is inserted instead of a normal nucleotide DNA replication stops at that point (chain termination) When this experiment is repeated with other dNTPs DNA molecules differing in length by 1 bp are created.

DNA sequencing cont.. These are then run on an electrophoresis gel Note: The smallest pieces at the start of the sequence are at the bottom. Therefore the sequence is read bottom to top Note that these are usually tagged with a radioactive sample. These can be compared.

DNA sequencing cont… Talk about newly automated methods – these involve tagging with a fluoresent dye. A computer gives a print out of the different bases.

Comparing Genomes Other genomes have been sequenced in conjunction with the human genome, and have continued. There are a large number of similarities between genomes. Genes found in a wide range of organisms are known as homologous genes. These are used to study function of genes.

Learning Activities Look at the arrangements document to clarify what information is required. Read DART pg 73 – 81. Read the Monograph pg 67 – 79 Scholar – 8 Internet research Worksheets