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5. How the Genome is Studied Maps and sequences Locus: location of a gene in a chromosome. Two genes are assorted (or segregated, i.e. are on the same chromosome) if an offspring has about 50% chance of inheriting both characteristics (deduced from the genes) from the same parent. Recombination: due to crossing-over (when cells divide) between chromosomes. If genes are closed together, there is a small chance of separation due to crossing-over. Genetic linkage map: constructed from observed segregation percentage, of certain characteristics. It shows the order and relative distance between genes (108 bp). 1 Maps and Sequences (cont.1) Physical map: tells the location of certain marquers (precisely known sequences) within105 - 106 base pair. Physical map construction is among the studied bioinformatics problems. Large-scale sequencing: done by breaking apart several copies of the piece to be sequenced (20 kbp) and by sequencing the (small) fragments directly. 2 Specific Techniques To obtain maps and sequences Produces nearly data that have errors (so algorithms are to be extended to handle errors. Virus and bacteria (organisms most used in genetic research) Virus consists of a protein cap (capsid) with DNA (or RNA) inside - cells starts producing-coded proteins which promotes viral DNA replication (new capsids break the cell membrane and attack other cells). - or DNA gets inserted into the host genome until... A bacterium (a single-cell organism having one chromosome, like Escherichia Coli) can multiply by simple DNA replication in very short period of time. 3 4 Cutting and breaking DNA Restriction enzyme: cut DNA at certain specific point (restriction site) Example: EcoRI cuts DNA at GATTC -between the G and the first A -the two strands, as GAATTC is a palindrome because GAATTC = GAATTC Endo(exo)-nuclease: cuts DNA internally (externally) Shotgun method breaks apart DNA molecules. Some fragments (issued from a purified DNA) are filtered and selected for cloning. Then they are sequenced (to determine the purified DNA's sequence) or they are collected to construct a cloning library. 5 6 Copying DNA (DNA Amplification) One molecule is not enough for experiences. DNA Cloning : by using nature itself, by inserting the initial piece in an organism (called host or vector) and by leaving the organism multiply itself. Afterwards, it is killed to produce DNA recombinant. Typical hosts: -Plasmid: piece of circular DNA which exists in bacteria. Limit of insert size: 15 kbp. -Phage (virus) : insert gets replicated when the virus infects a host colony. The size limit is 25 kbp, more with cosmids (40kbp, entire phage is replaced by the insert. 7 Copying DNA (Con.1) -YAC (Yeast Artificial Chromosome): artificial made chromosome where the insert lookslike an additional chromosome to the yeast replication mechanism (100-500kbp). BAC is for Bacterial Artificial Chromosome. 8 PCR PCR(Polymerase Chain Reaction): DNA polymerase is an enzyme that catalyses elongation of a single strand of DNA, provided there is template DNA to which this single strand is attached. The (small) double stranded DNA at the beginning is called a primer. Two steps are repeated : 1. Separation into two single strands by heat of the original double stranded ADN 2. Addition of primers and DNA polymerase action to produce a double strand. 9 Reading and measuring DNA Gel electrophoresis: a technique which is based on separation of molecules by their sizes by moving to a definite direction by action of an electric field, with speed inversely proportional to their size. Technique for reading: first obtain all fragments from the DNA molecules that end with an A (resp. T, G, C), then put them into four four different tubes, finally classify (by gel electrophoresis) the segments in each tubes according their size. Example: Given the DNA molecule GACTTAGATCAGGAAACT, the fragments which terminate by T are : GACT, GACTT, GACTTAGAT, GACTTAGATCAGGAAACT. 10 11 12 13