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Biochemistry 2280A
Chris Brandl

Sequencing Genomes - Huge impact on medicine and research- driving biology in many ways - “The inside story of how these bitter rivals mapped our DNA, the historic feat that changes medicine forever.” DNA and the Revolution In Personalized Medicine- pretty soon when you go to the doctor they will look at your genome sequence to properly prescribe you medicine. Every cancer differs dramatically- How the cancers differ will determine how you will be treated. Why Sequence Genomes 1. Identifies all of the genes that characterize an organism –the blueprint of life. (have all of the parts of the puzzle) 2. Allows comparative analyses between organisms. (confirmed the unity of life; helps to define function.) 3. Done within any individual species it may identify differences that result in disease. Sequencing the human genome has/will identify the genetic basis for many diseases. ($1000 genome 4. Identifies potential drug targets. (eg parasites may have unique genes and gene products that can be inhibited) Sequencing a genome involves. 1. Creating a Genomic DNA Library. 2. Doing many independent sequencing reactions. 3. Aligning the independent sequences into a continuous genomic sequence. And then try to annotate the genome. It’s not any more complicated in terms of features than we talked about before but the scale is enormous. A Genomic DNA library Defn: A collection of cloned DNA fragments representing all of the DNA in an organism’s genome. If we clone all the DNA from an organism into plasmid vectors, that would represent a genomic library for that organism. All of the clones represent all of the DNA in the genome. Assume we have the whole human genome, we cleave that with RE (this is the case where we have partial digest) to get millions of genomic fragments. Then, we insert all that fragments into plasmid vectors using DNA ligase, so we get representanents of each of those fragments in the population. Then that pop of clones is transformed into E.coli, which then makes up our genomic library. Fig. 10-11 Human DNA--- millions of genomic fragments Once you have the fragments you clone them into the plasmid vectors and you will get recombinant DNA molecules. Sequencing the H. influenza genome: Gram negative bacteria Genome aprox 2 million base pairs. To get the coverage of a full library: 2 million base pairs / 2000 bp/clone = 1000 clones minimum To be sure you have full coverage you would want an excess of several fold Creating the Genomic Library Here we have the bug, we extract the DNA from it. Sometimes we don’t want specific fragments so we can sonicate the DNA instead of using RE. We can purify those fragments in gel electrophoresis, and then select for those for example that are 2000bp in size. So the fragments are randomly being cut but are sorted according to size. We then take that DNA and clone it. We purify the DNA and now we prepare our library. In this case 20 000 clones are used (same as example ,we had fragments that are on average 2000bp so minimum of 1000 clones but we use 10-20 fold=20000). These are spotted onto grids so this is done in an automated fashion. Then, we randomly sequence all the clones we have. We get 25 000 independent sequences (12 million bp, and each sequence=400bp). Each of the 20 000 clones represents an independent part of the genome Sequence the ends of the genomic clones One clone Isolate plasmid DNA - Anneal primer - They got 25000 runs from 20000 clones and in total they got 12 million base pairs of DNA sequence. (aprox 6 fold excess in DNA sequence) Align the 25, 000 sequences into contigs First a computer searches for overlaps between the 25 000 sequence runs. Overlapping sequences are arranged in contigs. Aligning the independent sequences into a continuous genomic sequence Sequence CONTIG: A contiguous DNA sequence representing a portion of the genome. Sequence assembly is first done by computer searching, looking for overlaps Now we get to the part where we have to put everything back together. Once we have all the sequence fragments we have to align them into a continuous genomic sequence. It’s first done by computer looking for sequence overlap. It finds 2 sequences that over lap and stick them together to form part of the contig, shown in next slide. 20, 000 clones (25000 sequences representing 12 million bp and they can align that into 140 contigs. With 140 contigs you will have 140 gaps in a circular genome. We need to fill in the gaps to order the contigs and to complete the sequence. 2 types of gaps: sequence gaps and physical gaps. “Sequence gaps” are ones that can be closed by sequencing clones already present in the library. The computer looks for a clone DNA in which you have sequence to 2 different contigs. Showing 2 contigs that were put together simply through alignment. They know they got clones in the pool with one of its sequence ends matching one contig, while the other side matches this contig. They know that this sequence overlaps (is found between in the gap between these contigs). - The sequence in the middle of that clone will complete the sequence in that gap. - Had to sequence the clone from both side to find the one that will fill in the gaps. Physical Gaps There can be major challenges in fitting together the independent sequences. What about the gaps? The other type of gaps. Some sequences will not be represented in the original library resulting in gaps in the sequence, which means we often have to construct another library using a slightly different protocol in order to get those clones. Two strategies are often used; one is based on a probing hybridization type strategy. Another is based on PCR strategy. These generally represent sequences that
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