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Microbial Genomes 1) Methods for Studying Microbial Genomes 2) Analysis and Interpretation of Whole Genome Sequences

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  • Microbial Genomes 1) Methods for Studying Microbial Genomes 2) Analysis and Interpretation of Whole Genome Sequences
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  • Methods for Studying Microbial Genomes Why study genomes? History of genome sequencing Methods, Principles & Approaches
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  • Why study microbial genomes? until whole genome analysis became viable, life sciences have been based on a reductionist principle dissecting cell and systems into fundamental components for further study studies on whole genomes and whole genome sequences in particular give us a complete genomic blueprint for an organism we can now begin to examine how all of these parts operate cooperatively to influence the activities and behavior of an entire organism a complete understanding of the biology of an organism microbes provide an excellent starting point for studies of this type as they have a relatively simple genomic structure compared to higher, multicellular organisms studies on microbial genomes may provide crucial starting points for the understanding of the genomics of higher organisms
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  • Why study microbial genomes? analysis of whole microbial genomes also provides insight into microbial evolution and diversity beyond single protein or gene phylogenies in practical terms analysis of whole microbial genomes is also a powerful tool in identifying new applications in for biotechnology and new approaches to the treatment and control of pathogenic organisms
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  • History of microbial genome sequencing 1977 - first complete genome to be sequenced was bacteriophage X174 - 5386 bp first genome to be sequenced using random DNA fragments - Bacteriophage - 48502 bp 1986 - mitochondrial (187 kb) and chloroplast (121 kb) genomes of Marchantia polymorpha sequenced early 90s - cytomegalovirus (229 kb) and Vaccinia (192 kb) genomes sequenced 1995 - first complete genome sequence from a free living organism - Haemophilus influenzae (1.83 Mb) late 1990s - many additional microbial genomes sequenced including Archaea (Methanococcus jannaschii - 1996) and Eukaryotes (Saccharomyces cerevisiae - 1996)
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  • Microbial genomes sequenced to date currently there are 32 complete, published microbial genomes 25 domain Bacteria, 5 Domain Archaea, 1 domain Eukarya (www.tigr.org) around 130 additional microbial genome and chromosome sequencing projects underway
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  • Laboratory tools for studying whole genomes conventional techniques for analysing DNA are designed for the analysis of small regions of whole genomes such as individual genes or operons many of the techniques used to study whole genomes are conventional molecular biology techniques adapted to operate effectively with DNA in a much larger size range
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  • Pulsed Field Gel Electrophoresis agarose gel electrophoresis is a fundamental technique in molecular biology but is generally unable to resolve fragments greater than 20 kilobases in size (whole microbial genomes are usually greater than 1000 kilobases in size) PFGE (pulsed field gel electrophoresis) is a adaptation of conventional agarose gel electrophoresis that allows extremely large DNA fragments to be resolved (up to megabase size fragments) essential technique for estimating the sizes of whole genomes/chromosomes prior to sequencing and is necessary for preparing large DNA fragments for large insert DNA cloning and analysis of subsequent clones also a commonly used and extremely powerful tool for genotyping and epidemiology studies for pathogenic microorganisms
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  • Principle of PFGE two factors influence DNA migration rates through conventional gels - charge differences between DNA fragments - molecular sieve effect of DNA pores DNA fragments normally travel through agarose pores as spherical coils, fragments greater than 20 kb in size form extended coils and therefore are not subjected to the molecular sieve effect the charge effect is countered by the proportionally increased friction applied to the molecules and therefore fragments greater than 20 kb do not resolve PFGE works by periodically altering the electric field orientation the large extended coil DNA fragments are forced to change orientation and size dependent separation is re-established because the time taken for the DNA to reorient is size dependent
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  • Principle of PFGE
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  • the most important factor in PFGE resolution is switching time, longer switching times generally lead to increased size of DNA fragments which can be resolved switching times are optimised for the expected size of the DNA being run on the PFGE gel switch time ramping increases the region of the gel in which DNA separation is linear with respect to size a number of different apparatus have been developed in order to generate this switching in electric fields however most commonly used in modern laboratories are FIGE (Field Inversion Gel Electrophoresis) and CHEF (Contour-Clamped Homogenous Electrophoresis)
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  • + ++ + + + + + - - - -- - - - Electric Field 1Electric Field 2 Switch Time CHEF
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  • Preparation of DNA for PFGE ideally a genomic DNA preparation that contains a high proportion of completely or almost completely intact genome copies would be suitable for PFGE conventional means of DNA preparation are unsuitable for PFGE as mechanical shearing and low-level nuclease activity will result in fragmented DNA with an average size much smaller than an entire microbial genome (usually less than 200 kb in size) the solution to this is to prepare genomic DNA from whole cells in a semisolid matrix (ie. agarose) that eliminates mechanical shearing a very high concentration of EDTA is also used at all times in order to eliminate all nuclease activity
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  • Preparation of DNA for PFGE Procedure 1) intact cells are mixed with molten LMT agarose and set in a mold forming agarose plugs 2) enzymes and detergents diffuse into the plugs and lyse cells 3) proteinase K diffuses into plugs and digests proteins 4) if necessary restriction digests are performed in plugs (extensive washing or PMSF treatment is required to remove proteinase K activity) 5) plugs are loaded directly onto PFGE and run
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  • Preparation of DNA for PFGE for restriction digests, conventional enzymes are unsuitable as they cut frequently on an entire genome sequence producing DNA fragments that are far too small rare cutter restriction endonucleases cut genomic DNA with far less frequency than conventional restriction enzymes such as HindIII, BamHI etc. many rare cutter REs have 6-bp (or longer) recognition sites eg. NotI GC GGCCGC in many cases the frequency of cutting is highly species dependent eg. BamHI will cut far less frequently on a low GC% genome when compared to a intermediate or high GC content genome suitable rare cutter enzymes therefore have to be determined experimentally for each new species being studied
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  • Large insert cloning vectors BACs and PACs DNA cloning is another technique fundamental to molecular biology that requires adaptation in order to be useful in studying DNA at a whole genome scale conventional plasmid derived cloning vectors are only able to reliably maintain inserts less than 20 kb in size there are a number of approaches to generating clones with inserts in an intermediate size range (20 80 kB) such as cosmids, etc. the most commonly used vectors for cloning extremely large DNA inserts are BACs (Bacterial Artificial Chromosomes) and PACs (P1- derived Artificial Chromosomes) both BAC and PAC vectors are plasmid derived vectors distinguished from conventional vectors by extremely tightly controlled low copy numbers
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  • Large insert cloning vectors BACs and PACs these very low copy numbers help to limit the strain on host cellular resources generated by very large DNA inserts thus eliminating the rejection of large insert clones low copy numbers also help to limit recombination events with host genomic DNA BAC and PAC vectors both utilise E. coli as the host organism BAC vectors are based on the E. coli single copy F-factor plasmid the F-factor origin of replication is very tightly controlled PAC vectors are based on an identical principle but instead use a single copy origin of replication derived from P1 phage PAC vectors also contain a pUC19 cassette for improved vector purification
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  • Approaches to whole microbial genome sequencing aim of microbial genome sequencing projects is to construct, from 500 800 bp sequencing reads containing about 1% mistakes, a genome sequence of several megabases with an error rate lower than 1 per 10000 nucleotides with improving software, decreasing computation costs and advancements in automated DNA sequencing, an entire microbial genome project can be completed in a small laboratory in 1-2 years there are two main approaches to sequencing microbial genomes the ordered clone approach and direct shotgun sequencing both require both large and small insert genomic DNA libraries in order to be effective
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  • Approaches to whole microbial genome sequencing
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  • Ordered Clone Approach essentially this technique involves constructing a map of overlapping large insert clones covering the whole genome and then completely sequencing the minimum subset of these ordered clones there are a number of methods used

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