lecture 15 – gene cloning based on chapter 08 - genomics: the mapping and sequencing of genomes...
TRANSCRIPT
Lecture 15 – Gene CloningBased 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. 08.19 Dideoxy DNA sequencing of a theoretical DNA fragment
A G C T
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 10.2. The direct shotgun approach to obtaining the genomic DNA sequence of an organism.
Genome Assembly and Gene PredictionFEATURES Location/Qualifiers
source 1..9965640
/organism="Candidatus Solibacter usitatus Ellin6076" /mol_type="genomic DNA"
/strain="Ellin6076" /db_xref="taxon:234267"
gene 89..1402
/locus_tag="Acid_0001"
gene 1637..2755 /locus_tag="Acid_0002"
gene 2781..5228
/locus_tag="Acid_0003"
gene complement(5232..6827)
/locus_tag="Acid_0004"
gene complement(6844..8496)
/locus_tag="Acid_0005"
gene 8627..9400 /
locus_tag="Acid_0006"
gene 9397..10986
/locus_tag="Acid_0007"
gene 10983..11723
/locus_tag="Acid_0008"
gene 12211..12804
/locus_tag="Acid_0009"
gene 12823..12996
/locus_tag="Acid_0010"
gene complement(13323..13550)
/locus_tag="Acid_0011“
gene complement(13560..17015)
/locus_tag="Acid_0012"
gene complement(17148..19577)
/locus_tag="Acid_0013"