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CORNELL BIOMG 3320 - Genome Sequencing

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BioMG 3320 1st Edition Lecture 4Outline of Past LectureI. Solving the Genetic CodeII. Experiments to Determine Genetic CodeIII. More on the Genetic CodeIV. Degeneracy in the CodeOutline of Current LectureI. Comparing Genomes II. History of Genome SequencingIII. Gel Electrophoresis of Nucleic AcidsIV. DNA Sequencing: Gilbert MethodV. DNA Sequencing: Sanger MethodVI. Illumina SequencingCurrent LectureI. Comparing Genomes- The human genome consists primarily of introns (30% of genome). Exons, which are the parts that are put together to make mature mRNA are present in much smaller amounts- E.coli have a very gene-dense genome; the genes are very close together with very smallgaps- Single- cell eukaryotes have a genome that is also very gene-dense with small gaps between them. Introns are visibleThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Multicellular eukaryotes have a much less gene-dense genome. There are many introns and the gaps between the genes are bigger. Transposons are also present- Human genome has lots of introns, splicing, repetitive DNA. The genes are much more spread out- Plants have very large genomes; much larger than humans. There is not a lot of difference in the number of genesII. History of Genome Sequencing- First bacterial genome was discovered in 1995- First human genome was discovered in 2001- In the past, it took years to uncover one genome. However, now there is new technologythat allows us to decode genomes much faster- It is important to sequence DNA genomes for multiple reasons:-Allows you to find genes-Evolution: compare genomes of different species-Find functional information in sequences: line up different genomes and look for similarregions -Find mutations-Biotech and agriculture: find new, useful genesIII. Gel Electrophoresis of Nucleic Acids- Separation of nucleic acids by application of a voltage difference across a gel matrix- Charge on nucleic acids (neg charge on phosphate backbone) results in mobility within electrical field: from negative to positive terminal- The uniform structure of linear nucleic acids results in fractionation based on sized (longer molecules migrate more slowly)IV. DNA Sequencing: Gilbert Method- Gilbert’s method of encoding a genome consists of sequencing by chemical fragmentation; sequencing DNA by fragmenting in a specific way- The method is as follows:-Take sample of DNA-Prepare homogenous single-strand of DNA-Add 32P at 5’ phosphate end-Cleavage at specific nucleotides-Run electrophoresis, separating by size-RadioautographyV. DNA Sequencing: Sanger Method- DNA replication consists of adding complementary nucleotides to a template strand of DNA with DNA polymerase and a primer- Sanger’s method of encoding genome is by sequencing by polymerization-Need DNA template, primer, dNTPs, single ddNTP (modified substrates which lack 3’ hydroxyl), polymerase-DNA is copied by DNA polymerase from a DNA template starting from a fixed point-DNA polymerase incorporates ddNTP at end of a growing polynucleotide chain; once incorporated, the lack of the 3’ hydroxyl group prevents the addition of further nucleotides, causing elongation to terminate-Will generate multiple fragments with the same 5’ end, but differing in their lengths and3’ ends-The length of the fragments specifies the position of a specific nucleotide-The fragments labeled with radioactive primer that has been tagged with a fluorescent adduct-Fragments are separated by electrophoresis VI. Illumina Sequencing- This is how modern genome sequencing is done- Primers are covalently attached to surface, PCR amplifies single molecules of DNA producing PCR colonies- This is like Sanger’s method, but multiple DNA are being encoded simultaneously - The method is as follows:-All the DNA has the same red and blue sequences on top and bottom, but different middle sequences-Have a glass plate which DNA can covalently attach to-Use PCR on glass plate, amplifying so that millions of DNA molecules on plate-Use Sanger’s method of inducing chain terminating nucleotides with fluorescent dye-Wash, four-color imaging-Cleave dye and terminating groups, wash-Repeat- This is used to find disease-causing


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