Gene mapping and cloning of disease gene

1. Mapping - determining the location of elements within a genome, with
respect to identifiable landmarks.
Types of mapping…with tools/resources utilized
 Genetic mapping – linear description of DNA markers/genes on a
given chromosome with closely placed markers being inherited
together more often.
• linkage mapping
• pedigree
• polymorphic markers
 Physical mapping – physical location on the chromosome,
relating more towards exact positioning of gene elements.
• cytogenetic mapping
• somatic cell mapping
• radiation hybrid mapping
• restriction mapping - PFGE, BAC contigs, sequencing
 Comparative mapping
• gene sequences
• databases
• DNA chips
Gene Mapping
GENOME / GENETIC MAPS - Graphic representation of the relative positions
of genes on a DNA sequences.

2. If two alleles are far apart on the chromosome, as it is more likely
that a cross-over will occur between them and they will be separated.
Genes inherited in this way are said to be linked, and are referred to
as "linkage groups." For example, in fruit flies the genes affecting eye
color are inherited together because they appear on the same
chromosome.
Image of crossing over
occuring in
chromosomes
Sturtevant from the analyze of the fruit fly
genome found, for example, that leg length
was inherited with eye color more often than
with wing length, and that wing length was
inherited with eye color more often than with
leg length. .
Thus, he concluded, the gene for eye color must
be between the genes for wing length and leg
length.
.
CLASSICAL EXPERIMENT OF MAPPING

3. Linkage map versus physical map
It follows that a linkage map is a chromosome shows the
position of its known genes and/or markers relative to
each other in terms of recombination frequency, rather
than as specific physical distance along each
chromosome as in physical maps

4. GENETIC MARKER (Landmarks on the map) - Any inherited physical or molecular
characteristic that differs among individuals and is easily detectable in the laboratory is
a potential genetic marker
e.g. polymorphic/morphological markers like scorable phenotypes - in hemophilia,
wrinkled pea and pedigrees used for genetic map construction.
e.g. physical markers like RFLPs, RAPDs, AFLPs, VNTRs, STSs– used for physical map
construction

5. Approaches to Genetic mapping
1. Experimental crosses
- Backcrosses
- F2 intercrosses
- Introgression lines
- Recombinant inbred (RI) lines
2. Pedigree analysis
3. Association studies (Linkage disequilibrium, LD
mapping)
- With candidate genes (direct approach)
- Localized association studies (chromosomal region)
- Whole-genome association studies

6. CONSTRUCTING GENETIC LINKAGE MAPS
A. GENERATE MARKER DATA -
informative markers - polymorphic
and a population with known
relationships
B. CALCULATE ALL PAIR-WISE
DISTANCES – Test for linkage
between “close” markers, check for
recombinations.
C. GROUP MARKERS INTO
LINKAGE GROUPS - Unit of
distance in genetic maps =
centimorgans, cM
D. ORDER MARKERS IN EACH
LINKAGE GROUP - 1 cM = 1%
chance of recombination between
markers

7. GENETIC LINKAGE ANALYSIS

10. Constructing a Linkage map
 Two markers located in close
proximity on the same chromosome
tend to be linked
 Frequency of such occurrence leads
to distance estimation between markers
Thus the greater the frequency of
recombination (segregation, localization
of different chromosomes) between two
genetic markers, the farther apart they
are assumed to be. Conversely, the
higher the frequency of association
between the markers, the smaller the
physical distance between them.

12. The basic idea in pedigree mapping is to follow affected individuals and markers in
related families. Markers that are co-inherited with disease status are linked to the
causative gene. Most useful pedigrees contain– nil parentage error, large population
study, subsequent follow-up progeny study.
Mapping with Pedigrees
• Two unrelated pedigrees
• Haplotypes in each pedigree
are identified by numbers
(different for each family),
and determined from five
linked markers
• Dark blue - affected
• White - unaffected
• Light blue - status uncertain
• In each case one haplotype, in
red, co-segregates with the
disease

13. The resolution of mapping using crosses or pedigrees depends on the amount of
recombination, which is determined mostly by the number of meioses. Since
mapping studies cannot be easily performed with very large numbers of
individuals or very many generations, their resolution is generally poor. They
provide a good way for localizing genes to genomic regions, but typically do not
provide sufficient resolution to locate the gene. They are also inefficient at
finding alleles of very weak effect or at low frequencies in populations
One solution is to take advantage of historical recombination in the ancestry of a
lineage. Linkage disequilibrium is the non-random association of alleles at
different loci. These associations are caused by the mutational process and are
broken down by recombination in the population over time. LD mapping is
more efficient for finding genes of small effect (as may underlie most complex
diseases), for detecting contributions of rare alleles, and for localizing the gene of
interest.

15. Because map distances are additive, calculation of the A–B and A–C distances
leaves us with the two possibilities shown for the B–C distance.

16. The goal of genetic mapping is to find the genes underlying traits of interest.
The basic idea in all mapping studies is to look for markers that are inherited
with the trait. Physical linkage will result in co-inheritance, while recombination
and independent assortment will disrupt these associations. In general, markers
that are co-inherited with a trait are more likely to be physically close to the
genes underlying the trait.
Inheritance can be followed in controlled crosses, in pedigrees, or in the gene
genealogies of a species.
Traits can be simple (controlled by one or a few genes, with little environmental
influence; e.g. eye color in Drosophila, cystic fibrosis in humans) or complex
(controlled by many genes with many environmental influences; e.g. asthma,
heart disease).
Mendelian traits - discrete, controlled by one or a few genes
Quantitative traits - continuous, controlled by many genes
(QTL - Quantitative Trait Loci)
Mapping Quantitative Traits

18. PHYSICAL MAPPING - Cytogenetic Mapping
Cytogenetic mapping refers to observing a map location in
reference to a chromosomal banding pattern.
e.g. In situ hybridization - allow a rough
determination of localization of fragments
of DNA, but to not yield a direct measure
of distance.

19. Somatic Cell Hybrid Mapping
Somatic cell mapping can be used to map an element to a portion of a genome.
typically with chromosome resolution
Exploits the ability of rodent (hamster) cells to stably integrate genetic material from
other species.
Cells from the target genome are fused with hamster cells. The resulting cells are
then screened for cells (hybrids) that have retained one or more of the chromosomes
from the target genome.
Ideally, a complete set of hybrids can be constructed such that each has retained a
single chromosome from the target genome.
Using PCR and gene primers-DNA from the clones can be used to see if certain
genes are present in marked clones– leading to cytogenetic gene mapping.
DNA from cell lines used to form a panel and here no polymorphisms needed.
Finer mapping (higher resolution) can be obtained if hybrids are present in the panel
that contain partial chromosomes.
(E.g., translocations)

20. Somatic Cell Hybrid Mapping
Probe1
Probe2
Probe3
1 2 3 4 5
Chromosome
0 1
1 1
1 1 1 1 1
000
000
Probe1 -- maps to chromosome 2
Probe2 -- maps to chromosomes 3 and 4 -- possible paralogs,
pseudogene, or low-copy repeat
Probe3 -- maps to all chromosomes -- possible high-copy repeat
or ribosomal genes

21. Radiation Hybrid Mapping
Radiation hybrid mapping
is a method for high-
resolution mapping.
Exploits the ability of
rodent cells (hamster cells)
to stably incorporate
genetic material from fused
cells (same as SCH).
Panel is created by breaking
the chromosome of interest
or whole genome into
several fragments by high
X- ray irradiation. Use PCR
to screen clones that are
analyzed for presence or
absence of markers.

22. Restriction Mapping
Restriction enzymes - cut DNA at specific sites
Ex. EcoRI cuts at GAATTC
sites are often palindromic
GAATTC
CTTAAG
may leave blunt ends or overlaps
GGCC GG CC
CCGG CC GG
GAATTC G AATTC
CTTAAG CTTAA G
Restriction maps show the relative location of a selection of restriction sites
along linear or circular DNA.
HindIII BamHI PstII
PstII BamHI
HindIII
EcoRI

23. Restriction Mapping
BglII BamHI PstI
BglII
+BamHI
BglII
+PstI
BamHI
+PstI
4.2
5.2
3.6 3.5
3.3
2.6
1.7 1.7
1.4 1.4
1.2 1.2
1.0 1.0
1.2
0.7 0.9
0.5
0.3 0.30.3
BglII BamHI PstI BglII PstI
0.3 0.7 2.6 0.9 0.5 1.2

32. .
Physical maps. the highest-resolution physical map is the complete
nucleotide sequence of the chromosomes.
the lowest-resolution physical map an ordered set of landmarks (or
markers) with the physical distances in DNA-base-pairs from one
landmark to another.
Landmark or "sequence-tagged site," (STS) is a unique DNA sequence—
one that is found in only one place in the genome—and is a few
hundred base pairs long.
Thus finding a piece of DNA with STS shows exactly where in the
genome that piece of DNA belongs.
One of the early goals of the Human Genome Project was to select and
map a set of STS markers such that there would be at least one STS in
each stretch of 100 kb of the genome.
Markers
DNA

33. Comparison of chromosomal, linkage, and physical maps on Chr 17.
The chromosomal and linkage maps extend over the entire chromosome
with lines connecting the relative positions of loci mapped in both. An
example of a long-range physical map for a 1,200 kb genomic interval is
shown along with a short-range physical map for the gene itself.

34. Can be very useful in utilizing
animal models of human
disease, and also in exploring
the causes of complex diseases.
Comparing gene content,
localization and ordering
among multiple species.
Comparative Mapping
Sources of Information
sequence
mapping
BLAST
potential
orthologs
colocalization
sequence
mapping
Putative orthologs and
syntenic segments
GeneMap 99 (human)
• 42,000 ESTS
• 12,500 genes
Mouse RH consortium (mouse)
• 14,000 ESTs
UIowa EST placements (rat)
• 13,793 ESTs

37. Summary of Mapping Strategies

38. Genetic Variation Program supports research aimed at:
FIND differences in the sequence of DNA among individuals
are called genetic variation, which could explain some of the
differences among people, such as eye color and blood group,
and a risk for getting particular diseases.
DISCOVER single nucleotide polymorphisms (SNPs) and
other forms of genetic variation on a large scale across the
genome.
DEVELOP high-resolution maps of genetic variation and
haplotypes.
USE statistical methods to relate genetic variation to
phenotype.

39. Genome variations include mutations and polymorphisms.
If a given variation …A C T G A… …A C C G A… (T ⇔ C) is present
in less than 1 percent of people (for instance, 99.9 percent of people
have a T and 0.1 percent have a G), then the variation is called a
mutation, if in more than 1 percent then the variation is called a
polymorphism.
The term mutation was originally used by Dutch botanist Hugo De
Vries (1848–1935) to describe rapid changes in phenotype from one
generation to the next. Now the term mutation used to describe
heritable physical changes to genes.
Mutations range from the single base point mutations to mutations
that can span many functional genes.
About 90 percent of human genome variation comes in the form of
single nucleotide polymorphisms, or SNPs (“snip”)

40. The most popular SNPs ? Two of every three SNPs involve the replacement of
cytosine (C) with thymine (T).
Where SNPs can occur ? in both coding (gene) and noncoding regions of the
genome.
Are SNPs evolutionarily stable ? SNPs are evolutionarily stable --not changing
much from generation to generation.
Why analysis of SNPs are important? It helps to understand people's widely
differing susceptibilities to common diseases such as heart disease, diabetes, and
various forms of cancer. This answer shows another difference between mutation
and SNP:
Mutations may be associated with inherited diseases (the Huntington's
disease mutation), but SNPs act in more subtle ways. SNPs don't cause
disease—they are risk factors for disease.

41. SNPs and Disease Diagnosis
SNPs are not responsible for a disease state, they serve as biological markers for
pinpointing a disease on the human genome map, because they are usually
located near a gene found to be associated with a certain disease.
Two steps to create a genetic test for screening for a disease.
1) identify the disease-causing gene;
2) compare DNA for SNP patterns of a group of individuals - Non-affected and
Affected by the disease. This type of comparison, called an "association study",
can detect differences between the SNP patterns of the two groups,
Then physicians can screen individuals for susceptibility to a disease just by
analyzing their DNA samples for specific SNP patterns.

42. •Close genes are usually inherited together.
•In some cases, recombination (crossover) may occur during meiosis, thus separating
genes / loci. Chromosomal crossoveris the exchange of genetic material between
homologous chromosomes during prophase 1 of meiosis. The larger the distance
between 2 genetic loci, the greater the chance that they will be separated by a
crossover. The relative distance between two genes can be calculated using the
offspring of an organism showing two linked genetic markers, and finding the
percentage of the offspring where the two loci are not found together. The higher the
percentage of the offspring that does not show both traits, the farther apart on the
chromosome the two genes are. By calculating the recombination frequency between
the 2 genes/ loci, the approximate distance between them can be determined.
•If the position of one gene/locus in the genome is known (marker locus), the second
gene’s approximate location can be predicted (estimated).
•For example, in Drosophila the genes affecting eye color and wing length are inherited
together because they appear on the same chromosome.
•Genes are not considered reliable markers since they are not always close enough to
each other to allow precise determination of chromosomal location.
•Other physical markers (loci) are generally short stretches of DNA with known sequence
and location in the genome, such as single nucleotide polymorphisms (SNPs) and
microsatellites, which consist of repeating units of 2-6 bp.
Linkage Mapping
Linkage Mapping

43. Instead of beginning with a mutant organism and proceeding to identify the
mutated gene / gene function, reverse genetics approach starts with a
particular gene, mutating it, transferring it to cells or an organism then
analyzing its function.
•By studying encoded protein structure, inactivating mutations can be
designed so that it may interfere with protein functions.
•Truncation mutations; sequence alteration may be done by using different
fragments of the gene, which encode truncated gene products and analyze
the function of these constructs as compared to the full-length gene
product (wild-type).
•Site-directed mutations; the DNA sequence alteration involves a single
amino-acid change in the encoded product. It usually performed by PCR
using a primer(s) that contain the desired mutation and used to amplify the
normal gene in conditions that permits annealing of imperfectly matching
primer(s).
•Expression of mutated gene; altered DNA sequence is cloned into an
expression vector (under control of inducible promoter), transferred to
target cells (transformation / transfection) or an organism (transgenic
technology), allowed to be expressed (switched on or off on demand),
observe the effects and determine the gene product’s function.
Reverse Genetics