Transcript Gene Mapping - University of Delaware

Sequences,
Sequences,Sequences
and what they might mean
Comparative Genomics
 Gene
functions have been conserved
across evolution
 Nature solves a problem, it rarely solves it
again.
Eyeless Mutation
http://www.ucm.es/info/genetica/AVG/practicas/Drosophila/Drosophila.htm
Drosophila Eyeless
Homologous Genes
50% identical
Human Aniridia
Human gene expressed in correct location
in fly carrying the mutant eyeless gene
http://www.geo.de/GEO/fotografie/portfolio_des_monats/2001_10_portfolio_meckes/page2.html?SDSID=
Ectopic Expression of Human Eyeless Gene in Drosophila
http://as3.lib.byu.edu/~imaging/brad/set5/sl17.html
Flies with human eyeless homolog
do not develop “human” eyes
Develop compound eyes, even with
human homolog driving development
Fruit fly
Normal
Eyeless Gene
Developmental end
product depends on
context
Human
B
A
Normal
Eyeless Gene
D
E
Evolution & Genes

The function of many genes is conserved across
immense evolutionary distances.

The function of a gene is affected by its environment.

Many gene products function as part of a cascade or
pathway.
“Nothing in Biology Makes Sense Except in the Light
of Evolution” (Theodosius Dobzhansky)
Map Genes in the Context of
Chromosomes
Uses of Gene Mapping
 Identify


Heritable diseases
Cancer
 Identify



genes responsible for diseases.
genes responsible for traits.
Plants or Animals
Disease resistance
Meat or Milk Production
Types of Maps
 Nucleotide

Sequence Maps
complete or partially sequenced organisms
 Cytogenetic


Breakpoints in disease
Direct binding of probes to chromosome
 Genetic

Maps
Linkage Maps
Markers
 Physical
Maps
Mapping Genes
 Essential
element -- Markers
 Differences between two members of a
species.
 Typically between 1-400 nucleotides in
length.
 Can also be gross chromosomal
rearrangements.
DNA Sequencing
 Two



methods originally developed:
Chemical or Maxim and Gilbert method.
Dideoxynucleotide or Sanger method.
Both Gilber and Sanger won Nobel prizes for
developing these techniques.
 Dideoxynucleotide
method is by far the
most common employed today.
Automated Sequencing
 Each
nucleotide is labeled with a
different fluorescent chromophore.
 Uses Taq polymerase to incorporate
fluorescent dideoxy nucleotide.
 Analysis on a polyacrylamide gel or
capillary matrix that is can separate
DNA based upon a 1 nucleotide
difference.
 30-1000 nucleotides in length can
theoretically be determined.
O
O P O
Base
CH2
Deoxy
O
O
OH
O
O P O
Dideoxy (3’2’)
O
Base
CH2
O
Copyright Cold Spring Harbor Laboratories
G A T C G G A T C C
G A T C G G A T C C
T A G G
G A T C G G A T C C
C C T A G G
dd
G A T C G G A T C C
C C T A G G
dd
G A T C G G A T C C
GC C T A G G
dd
Nucleotide Sequencing
 Complete

Genome Sequencing
Bacterial Artificial Chromosomes (BAC)
• Can grow in bacteria
• Have large inserts 100,000-300,000
nucleotides


Collect a large library of BACs
Restriction Endonuclease (RE) Map
• RE - cuts DNA at specific sites
• EcoRI cuts at GAATTC
B
A
C
s
meds.queensu.ca/~mbio318/ EXTRA_MATERIAL.html
Genomic Sequencing
 Once
BAC map complete
 Sequence ends of BAC clones.



With dense BAC map, large regions may be
covered by overlapping the sequences.
Individual BACs can be completely
sequenced.
Individual BACs can be hybridized to
chromosomes to identify chromosome of
origin.
Recombination
 Permits


Mapping by providing:
Linkage groups
Distances
 During
Meiosis
Recombination
 Linkage
Groups - markers that tend to
remain together.
 Distance - the further apart two markers
lie, the more often recombination will occur
between those markers.
 Markers on the same chromosome can be
so far apart that they appear in different
linkage groups.
Cytogenetic Mapping
Philadelphia Chromosome
 Chronic
myelogenous leukemia (CML) is
characterized by a reciprocal translocation
between chromosomes 9 and 22 that
produces the Philadelphia chromosome.
Invariably there is disease progression,
with loss of the capacity for terminal
differentiation by the hematopoietic stem
cell, resulting in an acute leukemia.
 www.clubstewart.co.uk/cml.htm
Genetic Linkage Maps
Polymorphism
 Polymorphisms




Polymorphism - an allele present in a
population that exhibits multiple forms.
Monomorph - Single form in a population.
Common.
Typically result in no effect on survival of
individual.
Types of Markers
 Single

Nucleotide Polymorphisms (SNPs)
Occur approximately every 100-300 bp in
humans.
Names: rs8111765
Markers
 Microsatellites


or Tandem Repeats
acgCACACAtgc
acgCACACACAtgc
Physical Maps
 Sequence


Tagged Sites (STSs)
Use Polymerase Chain Reaction (PCR).
Primers based upon:
• random sequence
• expressed sequence tag



Amplify from library of clones containing
large inserts (BAC).
Relate to BAC map.
If more than one on same clone, then close
together
Types of questions

Where does gene X exist within the genome of organism Y?
What are some flanking markers?

Which genes exist on a chromosome, and in what order do they
appear?

Show the genes that exist in region R of the chromosome. Show
me the corresponding sequence data for that region.

Display the region of a chromosome between points A and B.

Show both the cytogenetic and sequence map for that region,
aligned to each other based on markers that have been placed
on both maps.

What is the distance between two genes? (Note: scale depends
on the type of map on which those genes have been placed.)

I know the cytogenetic location of gene X. What is the
corresponding physical location?
http://www.ncbi.nlm.nih.gov/mapview/static/MapViewerHelp.html
Genes and Maps
 Bcl2
 Map
Link
 OMIM Link
 Syntenic Relationships - tendency of
closely linked genes to remain linked
during evolution.
GO evidence codes
Pathways

KEGG pathways
Examine Cell Adhesion Molecules
-What does JAM3 appear to interact with?
-What information is available if you click on
JAM2?
What motifs are found in JAM2?
Click on Tight Junction
-What molecules appear to interact with the JAMJAM dimer? What is ZO-1