Transcript Document

Chapter 12: Genomics
Fig. 12-1
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)
Overview of genome sequencing
Fig. 12-2
Sequencing the ends of clones in a library
Fig. 12-4
Overview of genome sequencing
Fig. 12-2
Fig. 12-5
Fig. 12-6
Overview of genome sequencing
Fig. 12-3
Fig. 12-7
Fig. 12-8
Several orders of magnitude resolution separates
cytogenetic from gene-level understanding
Fig. 12-9
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)
Inheritance of an RFLP:
Fig. 12-10
Inheritance of an RFLP:
Determining
linkage to a known gene
Fig. 12-10
Inheritance of an RFLP:
Determining
linkage to a known gene
Fig. 12-10
Linkage analysis of a gene and VNTR markers
Fig. 12-11
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)
Using a SSLP marker
to map a disease
Fig. 12-12
Using a SSLP marker
to map a disease
Unlinked
Linked to P
Linked to p
Unlinked
Fig. 12-12
Polymorphism markers
can provide a high
resolution map
Linkage map of
human chromosome 1
Fig. 12-13
High-resolution cytogenetic mapping
is based on:
• In situ hybridization: hybridization of known
sequences directly to chromosome preparations
• Rearrangement break mapping
• Radiation hybrid mapping
FISH analysis
using a probe
for a muscle
protein gene
Fig. 12-14
Survey clones from the region of the break
to determine one that spans the break
Fig. 12-16
Survey clones from the region of the break
to determine one that spans the break
FISH analysis locates the sequence
and the breakpoint cytogenetically
Fig. 12-16
Cytogenetic map
of human
chromosome 7
Fig. 12-24
Determining the sequence map sites of
rearrangement breakpoints and other mutations
Fig. 12-17
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)
Genes and their components
have characteristic sequences
Bioinformatic analysis of raw sequences
can suggest possible features
Fig. 12-19
Confirmation of genes and their architecture
is obtained by analysis of cDNAs
cDNA subprojects are key facets of a genome project
Fig. 12-20
High-resolution genomics arises through
the combination of bioinformatics and experimentation
Fig. 12-21
Using bioinformatics to make detailed gene predictions
Fig. 12-22
Complete sequence and partial interpretation
of a complete human chromosome
Fig. 12-23
Comparative genomics
reveals ancestral
chromosome
rearrangements
Fig. 12-26
Microarray analysis – a form of functional genomics
Arrays hybridized to cDNAs prepared from total RNA
Relative intensity (color-coded) reflects abundance of individual RNAs
1046 cDNA array
Fig. 12-27
65,000 oligo array
(representing 1641 genes)
Fig. 12-
Fig. 12-