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-