Lecture 15 – Gene Cloning Based on Chapter 08 - Genomics: The Mapping and Sequencing of Genomes Copyright © 2010 Pearson Education Inc.

1. Introduction

10. Artificial Chromosomes

13. Genomic Libraries

14. Size Selection of DNA Fragments using Agarose Gel Electrophoresis

Slide 15 - DNA Sequencing and Analysis of DNA Sequences

DNA Sequencing Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Fig Dideoxy DNA sequencing of a theoretical DNA fragment A G C T

Automated Sequencers

Pyrosequencing

Sequencing Genomes Structural Genome Biologists use two general approaches to sequencing whole genomes: The mapping approach – first used, slower, but informs you of problems. The Whole Genome“Shotgun” approach – rapid, but requires greater computational expertise. Works well on smaller genomes with low complexity.

Genome Sequencing Using a Mapping Approach Genetic mapping a.Cytogenetic maps b.FISH maps

Genome Sequencing Using a Mapping Approach Genetic mapping a.Cytogenetic maps b.FISH maps c.Radiation Hybrid maps Others to be discussed later a.RFLP maps b.SNP maps c.STR maps d.VNTR maps

Genome Sequencing Using a Mapping Approach Identify and genetically map marker genes. Break genome up into BAC or YAC clones Construct a Clone Contig Map Sequence each BAC or YAC and locate the markers in the sequences. Order the BACs or YACs to make a genomic sequence. a.Cytogenetic maps b.FISH maps c.Restriction maps d.Radiation hybrid maps e.Clone Contig maps

Genome Sequencing Using a Mapping Approach

Ultimately through the use of these techniques a high density physical map of sequence polymorphisms can be generated and used as the basis for squencing the genome. Genome Sequencing Using a Mapping Approach

Genome Sequencing Using a Whole-Genome Shotgun Approach 1.The shotgun approach obtains a genomic sequence by breaking the genome into overlapping fragments for cloning and sequencing. 2.A computer is then used to assemble the genomic sequence. 3.Advances that have made this approach practical for large genomes include: a.Better computer algorithms for assembling sequences. b.Automation in the actual sequencing. c.Faster automated sequencers that sequence millions of sequences at one time.

Genome Sequencing Using a Whole-Genome Shotgun Approach Figure The direct shotgun approach to obtaining the genomic DNA sequence of an organism.

Genome Assembly and Gene Prediction FEATURES Location/Qualifiers source /organism="Candidatus Solibacter usitatus Ellin6076" /mol_type="genomic DNA" /strain="Ellin6076" /db_xref="taxon:234267" genegene /locus_tag="Acid_0001" genegene /locus_tag="Acid_0002" genegene /locus_tag="Acid_0003" genegene complement( ) /locus_tag="Acid_0004" genegene complement( ) /locus_tag="Acid_0005" genegene / locus_tag="Acid_0006" genegene /locus_tag="Acid_0007" genegene /locus_tag="Acid_0008" genegene /locus_tag="Acid_0009" genegene /locus_tag="Acid_0010" genegene complement( ) /locus_tag="Acid_0011““ genegene complement( ) /locus_tag="Acid_0012" genegene complement( ) /locus_tag="Acid_0013"