03 - January 15, 2013.docx

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University of Toronto St. George
Molecular Genetics and Microbiology
Johanna Rommens

Lecture notes – January 15, 2013 The human genome project – traits could in theory be mapped in genome; feasible to track genes inherited in Mendelian fashion; how useful complete maps of genome would be; difficult task to sequence; When genome project was first proposed – why not just sequence the protein-coding sequences? Take RNA convert to cDNA, sequence – allow functional analysis, which was the major interest Select model organisms – some already partially mapped – for example, c elegans Genome project purpose to have multi-purpose maps – understand genome Look for landmarks along chromosomes Local chromosome regions – sub region of chromosome – to get good detail of the map – merge chromosome maps together to get full picture Physical mapping tools Top four methods – broad resolution – poor resolution but looking at whole or portions of chromosomes Tools that came about whole chromosomes – Giemsa banding – Somatic cell hybrids – maintains a human chromosome in the DNA of the cell line (usually mouse, hamster) – chromosome may rearrange Banding patterns Giemsa banding standard for mapping chromosomes Start from top of shorter arm, position arm – go down chromosome Second group of chromosomes One interesting fact is that chromosome 1 is not bigger than 21 Chromosomes ordered in size by Giemsa banding – 21 shorter than 22 Patient with Downs syndrome Tools of physical mapping correlate with studies of human chromosomes When have sequence and cannot align it – when look at sequence, Use tool to map onto segment of chromosome – even if cannot align by sequence alignment Using hybridization in-situ By hybridization of a probe – detect complimentary sequence on chromosomes If have chromosome preparation – if hybridize with probe that is labelled fluorescently, hybridize onto slide – Modification – chromosome painting technique – probe represents full chromosome – probe set for each chromosome, hybridize to slide and see entire chromosome light up Looking at chromosomes Lose Gimsa staining with in situ 1p – tip of shorter arm 1q – tip of longer arm Chromosome has both probes towards bottom right corner Crude method – how do you know that the tip isn’t bent behind and thus hidden Interphase – circular region – in interphase, can see signal; if one green spot, expect two Limit to the resolution: one green spot may be two on top of each other One feature of this technique that makes this still used in present – tool that lets you look at a SINGLE CELL Spectral karyotype If you suspect that the cells are not equal – ex. Cancer !! Cancer growths are evolving cells –g enomes changing – not necessarily clonal, may have pockets of clonality Painting technique – a slide every chromosome painted with different colour; b is normal cell; c is cancer cell Parts of tumour may be differnet, have different makeup so f chromosome Approaches to physical mapping Another application is when making mouse model of disease- need to manipulate genome at particular locus – use reagents that are harsh to genome – use FISH to check that haven’t modified genome too much – to be sure that the genome is intact A large genome – resolution Clone fragments of DNA – take entire genome, smashing it, indepently pulling out each piece, generating library where have every single piece – have overlapping pieces with sufficient information so can generate the entire map back In yeast, called artificial chromsomes; bacteria, episomes or plasmids Generation of genoic li
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