Fig. 12-1
Chapter 12: Genomics
Genomics: the study of whole-genome structure, organization, and function
Structural genomics: the physical genome; whole genome mapping
Functional genomics: the proteome, expression patterns, networks
Creating a physical map of the genome
• Create a comprehensive genomic library (use a vector that incorporates huge fragments)
• Order the clones by identifying overlapping groups (e.g., sequencing ends to determine “contigs”)
• Sequence each contig
• Identify genes and chromosomal rearrangements within each contig (correlates the genetic and physical maps)
Fig. 12-2
Overview of genome sequencing
Fig. 12-4
Sequencing the ends of clones in a library
Fig. 12-2
Overview of genome sequencing
Fig. 12-5
Fig. 12-6
Fig. 12-3
Overview of genome sequencing
Fig. 12-7
Fig. 12-8
Fig. 12-9
Several orders of magnitude resolution separates cytogenetic from gene-level
understanding
Creating a high-resolution genetic map of the genome requires many “markers”
• Classic mutations and allelic variations (too few)
• Molecular polymorphisms; selectively neutral DNA sequence variations are common in genomes
Example: Restriction Fragment Length Polymorphisms(RFLP markers)
Fig. 12-10
Inheritance of an RFLP:
Fig. 12-10
Inheritance of an RFLP:
Determininglinkage to a known gene
Fig. 12-10
Inheritance of an RFLP:
Determininglinkage to a known gene
Fig. 12-11
Linkage analysis of a gene and VNTR markers
Creating a high-resolution genetic map of the genome requires many “markers”
• Classic mutations and allelic variations
• Molecular polymorphisms; selectively neutral DNA sequence variations are common in genomes
Example: Restriction Fragment Length Polymorphisms(RFLP markers)
Example: Simple Sequence Length Polymorphisms(SSLP markers)
SSLP: Simple sequence length polymorphism
• VNTR repeat clusters (minisatellite markers)
• dinucleotide repeats (microsatellite markers)
VNTRs can be detected by restriction/Southern blot analysis; both detected by PCR using primers for each end of the repeat tract
Variable number tandem repeats (VNTRs)
• “minisatellite” DNA
• 15-100 bp units; repeated in 1-5 kb blocks
• expansion/contraction of the block due to meiotic unequal crossingover
• crossingover so frequent that each individual has unique pattern (revealed by genomic Southern blot/hybridization analysis)
Fig. 12-12
Using a SSLP markerto map a disease
Fig. 12-12
Using a SSLP markerto map a disease
UnlinkedLinked to PLinked to p
Unlinked
Fig. 12-13
Polymorphism markerscan provide a highresolution map
Linkage map of human chromosome 1
High-resolution cytogenetic mapping is based on:
• In situ hybridization: hybridization of known sequences directly to chromosome preparations
• Rearrangement break mapping
• Radiation hybrid mapping
Fig. 12-14
FISH analysis using a probe for a muscle protein gene
Fig. 12-16
Survey clones from the region of the breakto determine one that spans the break
Fig. 12-16
FISH analysis locates the sequenceand the breakpoint cytogenetically
Survey clones from the region of the breakto determine one that spans the break
Fig. 12-24
Cytogenetic map of human chromosome
7
Fig. 12-17
Determining the sequence map sites ofrearrangement breakpoints and other mutations
Mapping & determining a gene of interest
Fig. 12-18
Genome sequencing projects
• Sequence individual clones and subclones (extensive use of robotics)
• Identify overlaps to assemble sequence contigs (extensive use of computer-assisted analysis)
• Identify putative genes by identifying open reading frames, consensus sequences and other bioinformatic tools
Once a genomic sequence is obtained, it is subjected to bioinformatic analysis to determine structure and function
• Identify apparent ORFs and consensus regulatory sequences to identify potential genes
• Identify corresponding cDNA (and EST) sequences to identify genuine coding regions
• Polypeptide similarity analysis (similarity to polypeptides encoded in other genomes)
Fig. 12-19
Genes and their components
have characteristic sequences
Bioinformatic analysis of raw sequencescan suggest possible features
Fig. 12-20
Confirmation of genes and their architectureis obtained by analysis of cDNAs
cDNA subprojects are key facets of a genome project
Fig. 12-21
High-resolution genomics arises throughthe combination of bioinformatics and experimentation
Fig. 12-22
Using bioinformatics to make detailed gene predictions
Fig. 12-23
Complete sequence and partial interpretationof a complete human chromosome
Fig. 12-26
Comparative genomics reveals ancestral
chromosome rearrangements
Fig. 12-27
Microarray analysis – a form of functional genomics
1046 cDNA array 65,000 oligo array(representing 1641 genes)
Arrays hybridized to cDNAs prepared from total RNARelative intensity (color-coded) reflects abundance of individual RNAs
Fig. 12-
Fig. 12-