BSCI222 Lecture 18 11 7 13 Chapter 20 Genomics and Proteomics Sequencing the Human Genome Boyer and Cohen restriction enzymes and transformation methods were able to take DNA cut it with restriction enzymes as well as cutting a plasmid vector and then ligate pieces of DNA into the vector and then put it into E coli and have many copies of that genome then cloned o Restriction enzymes are isolated from various species of bacteria where they normally protect it from invading viruses They respond a very specific sequence that is not found in the bacterial genome but is in the viral genome Name after genus and species of the bacteria Cut at palindromes same order on both strands reading 5 to 3 The enzyme cuts the DNA backbone between the genes bind as dimer so do a symmetrical cut o DNA molecules cut with the same restriction enzyme have complementary sticky ends that pair if fragments are mixed together The nicks in the sugar phosphate backbone of the two fragments can be sealed by DNA ligase definition of recombinant DNA o One of the first uses of this technology was to clone pieces of a genome Cloning is necessary because for sequencing and studying helps to have many copies at once Method for making a genomic library o If you only add a little bit of restriction enzymes not every restriction site will be cut in every copy of the molecule might skip sites etc End up with overlapping DNA fragments if you have a limited amount of restriction enzymes Each of the fragments can be recombined with a plasmid DNA vector circular from a bacterium called a cloning vector When the bacteria has been grown out on a plate into colonies some of the clones will have all of the gene of interest others will have parts of it and most will have none of it To find the gene of interest have to screen the libraries with radioactively labeled probes Put all the bacteria on plate let each single cell grow into a colony place filter paper on the plate it picks up some bacterial cells replicates the pattern of colonies on it wet the paper with a solution that lyses the cells and the DNA is released DNA immediately binds to the paper because of the charges Next find the particular colony that carries the gene of interest you must already know something about the gene maybe from a different organism and you label it with p 32 radioactive Some of the colonies end up with the probe attached to the DNA on the filter then put X ray film on top as p 32 decays exposes the film makes spots This reveals which bacterial colonies have the DNA of interest o Summary genomic DNA cut with restriction enzyme ligated to vector transfer to E coli grow vat of each clone search library of clones with p 32 labeled probe The Boyer Cohen patent immediately facilitated mapping the human genome o Human genetics was really lagging bad for genetic mapping little offspring can t force people to mate in a certain way few genetic markers o New markers RFLP restriction fragment length polymorphisms markers polymorphisms in the restriction sites Genotyped by developing a clone for a particular part of the genome making it radioactive and then using it as a probe Essentially missing a restriction site so one person s DNA will be cut into say 3 pieces while another s only into 2 because they only have one real restriction site there instead of 2 A mutation creates a polymorphism Some copies have both restriction sites and others only have one o Human genome 3x10 9 base pairs o HaeIII 4bp cutter has 4bp recognition sequence recognizes GGCC When DNA from two persons is digested by HaeIII two different patterns appear on the autoradiograph of the gel because one s chromosomes have more restriction sites than others Essentially cuts the genome every 256 bases gives you about 10 7 fragments 10 000 000 See a great big smear on the gel not a single band of 256 bases because it does not cut EXACTLY every 256 bases just on average End up with a distribution of fragments looks like a smear Have to probe the gel with just the piece of DNA that interested in clone and make radioactive can visualize where those bands are in the gel smear Put filter paper under the gel with wet sponges on top and paper towels under the filter paper water draws through brings DNA with it which gets stuck covalently bonded sticking up and ready to H bond to any probe you want to put on it on the nylon filter paper o For each radioactive probe can sequence from the human genome and it becomes a marker o This was done for years analyzing the human genome for restriction polymorphisms RFLP markers Essentially an unlimited amount of markers could start mapping human genetic diseases o Interestingly when comparing recombination between males and females there s a big difference a lot more recombination in female myosis than in male myosis longer by about 50 even though it s the same DNA o One of the first ones was cystic fibrosis Mendellian disease Causes thick mucus in the lungs Happens in about 1 3000 births relatively high frequency genetic disease o Looked in the population for statistical associations with fragment differences and the disease Found a bunch of markers that showed a statistical association Mapping genetic diseases o Next step go out and gather pedigrees of families that are segregating for this disease Have to get a large amount of families because humans don t have very large families Started with 2 markers mapped the distance between them on chromosome 7 called labs around the world to find other markers probes within this region linkage studies Want to find markers that are on either side of the cystic fibrosis gene to find it o Then have to sequence the region At the time had to do chromosome walking start at a marker and then from the genomic library identify overlapping clones until you are sure you have gone past the gene of interest By making probes complementary to areas of overlap between cloned fragments in a genomic library connect the gene of interest to a previously mapped linked gene Each step takes about a month If lucky have about 1 marker cM or 1 marker Mb Typically clone is about a thousand steps long a thousand walking steps a thousand months to get across one cM of the genome o Had to find a better way Chromosome jumping Take a piece of the genome cut a larger piece out and then ligate it together to make a circle Then re clone the region where the ends came together which were originally about 80 150bp apart but now are right next to