Gene Mapping

Dr.T.B.Nyambo
1st November 2005
 The Human Genome Project(HGP)
The 3bn project formally started in 1990 initially headed by James Watson
till1992.The U.S. Human Genome Project was a 13-year effort coordinated
by the U.S. Department of Energy and the National Institutes of Health. The
project originally was planned to last 15 years, but rapid technological
advances accelerated the completion date to 14th April2003. The project did
not study all of the DNA found in human cells; some heterochromatic areas
(about 8% of the total of 3 billion bp in haploid cell) remain un-sequenced. In
1998 Celera Genomics HGP, a private company started to sequence with
intention to patent.

Project goals were to.

identify all the approximately 20,000-25,000 genes in human DNA.
determine the sequences of the 3 billion chemical base pairs that make up
human DNA,
store this information in databases,
improve tools for data analysis,
transfer related technologies to the private sector, and
address the ethical, legal, and social issues (ELSI) that may arise from the
project.
 Practical benefits to learning about DNA

Knowledge about the effects of DNA variations

among individuals can lead to revolutionary
new ways to diagnose, treat, and prevent the
human disorders.
Besides providing clues to understanding
human biology, learning about nonhuman
organisms' DNA sequences can lead to an
understanding of their natural capabilities that
can be applied toward solving challenges in
health care, agriculture, energy production,
environmental remediation, and carbon
sequestration.
 Gene mapping
"Gene mapping" refers to the mapping of genes
to specific locations on chromosomes. It is a
critical step in the understanding of genetic
diseases. There are two types of gene mapping:
Genetic Mapping - using linkage analysis to
determine the relative position between two
genes on a chromosome.
Physical Mapping - using all available
techniques or information to determine the
absolute position of a gene on a chromosome
 Genetic map
A genetic map is a representation of the genes on a
chromosome arrayed in linear order with distances
between loci expressed as percent recombination
(map units, centimorgans). Also called a linkage
map.
A. Genes on the same chromosome are described as
linked or syntenic.
1. Humans have 24 linkage groups, corresponding to
the 22 autosomes, plus X and Y chromosomes. 2.
Groups of genes that are widely separated on a
chromosome may show independent assortment
B. The genes on a chromosome can be represented
as a single linear structure that goes from one end of
the chromosome to the other.
 Genetic Distance
Genetic distance is measured by frequency of crossing over
between loci on the same chromosome.
A. One map unit = one centimorgan (cM) = 1% recombination
between loci.
B. The farther apart two loci are, the more likely that a crossover
will occur between them. Conversely, if two loci are close
together, a crossover is less likely to occur between them.
C. Recombination can only be detected between two loci, both of
which are heterozygous.
1. The dominant/recessive relationships must allow for
detection of recombinants.
2. The most useful systems involve codominant alleles.
3. Efficient mapping requires polymorphic loci, i.e. loci with two
or more common alleles. Loci that have a single common allele
are described as monomorphic.
4. Any variations in DNA, whether in coding regions of genes or
in noncoding regions, can be used as genetic markers.
 There are several levels of detail
Maps of DNA can have several levels of detail; from the
banding patterns of the chromosomes, to clones of
overlapping segments of DNA, and ultimately to the
base-by-base sequence of DNA
 CF
The ultimate goal of gene mapping is to clone
genes, especially disease genes. Once a
gene is cloned, we can determine its DNA
sequence and study its protein product.
In 1985, the CF gene was mapped to
chromosome 7q31-q32 by linkage analysis.
Four years later, it was cloned by Francis
Collins and his co-workers.
We now know that the disease is caused by
the defect of a chloride channel
 Linkage analysis

The genetic mapping is based on the linkage

between "loci" (locations of genes). If two loci
are usually inherited together, they are said to
be "linked".
Two loci on different chromosomes are not
linked, because they are usually separated by
independent assortment.
Crossing over can link genes
Crossing over between two homologous chromosomes
 Linkage analysis

A locus (singular of loci) may have different

sequences, referred to as alleles.
Consider two loci A and B, each having
two alleles (one from mother, another from
father). A1 and A2 are the two alleles of
locus A ; B1 and B2 are the two alleles of
locus B. Initially, A1 and B1 are located on
the same chromosome. A2 and B2 are
located on a different chromosome.
 Linkage analysis

.
 Linkage analysis
The DNA crossover may cause recombination
of loci A and B. Namely, A1 and B2 (or A2 and
B1) are located on the same chromosome.
The recombination frequency depends on the
distance between the two loci and the position
of crossover (the chiasma).
The closer they are, the less likely the
recombination will occur
 Mapping human genes by using humanrodent somatic cell hybrids

Cell fussion is done by using Sendai virus

The selective technique involves the
application of a chemical, aminopterin,
that blocks the de novo synthetic pathway,
confining DNA synthesis to the salvage
pathway.
Two essential salvage enzymes, thymidine
kinase (TK) and hypoxanthine-guanine
phosphoribosyl transferase (HGPRT),
.
 Mapping human genes by using humanrodent somatic cell hybrids

Because each is deficient for one enzyme,

neither the mouse nor the human cells are able
to make DNA individually.
In the hybrid cells, however, the tk+ allele
complements the hgprt+ allele, so the cells can
make both enzymes. Therefore, DNA is
synthesized and the cells can proliferate.
 Somatic cell hybridization
Most human chromosomes are eliminated from
the hybrid cell cultures because their loss has
no effect on the cultures' ability to grow.
But, to continue to grow in medium containing
hypoxanthine, aminopterin, and thymidine (HAT
medium), a hybrid culture must retain at least
one of the human chromosomes that carries
the tk+ allele.
 Flow cytometry
Human chromosomes show considerable variation in
size and DNA content . In addition, the base
composition can vary considerably: chromosomes
that are known to be gene-rich or gene-poor have a
comparatively high % (G + C) or % (A + T)
respectively.
For example, chromosomes 21 and 22 are similar in
size and in DNA content, but chromosome 21 is
gene-poor and has a relatively high % (A + T),
whereas chromosome 22 is gene-rich and has a
relatively high % (G + C).
As a result of the size differences and differences in
base composition, human chromosomes can be
separated by flow sorting (also called flow
cytometry)
 Gene mapping
This technique can be extended to obtain mapping
data. One extension of the hybrid cell technique
is called chromosome-mediated gene
transfer.
First, samples of individual human chromosomes
are isolated by fluorescence-activated
chromosome sorting (FACS) .
In this procedure metaphase chromosomes are
stained with two dyes, one of which binds to AT-
rich regions, and the other to GC-rich regions.
 Gene mapping

Cells are disrupted to liberate whole chromosomes into

liquid suspension.
This suspension is converted into a spray in which the
concentration of chromosomes is such that each spray
droplet contains one chromosome.
The spray passes through laser beams tuned to excite
the fluorescence.
Each chromosome produces its own characteristic
fluorescence signal, which is recognized electronically,
and two deflector plates direct the droplets containing
the specific chromosome needed into a collection tube.
Fluorescence-activated chromosome sorting
(FACS)

.
Cellular DNA is a compact molecule
The linear length of DNA in an average sized
human chromosome is about 5 cm, but it is
compacted by various hierarchies of folding in
metaphase chromosomes to a few microns
As a result, standard in situ hybridization
against the highly condensed chromatin of
metaphase and prometaphase chromosomes
does not have a high mapping resolution.
To obtain higher resolution, DNA probes are
hybridized to the naturally extended interphase
chromosomes or to artificially stretched
chromatin.
 Levels of DNA organization
.
 Molecular combing to stretch DNA
The principle of molecular combing is analogous to that of the receding sea,
which aligns algae on the beach.
In molecular combing, algae are replaced by linear macromolecules, like
DNA, which can be anchored on several surfaces by their extremities only,
provided the right physico-chemical conditions are used .
The front wave of the receding sea is here replaced by a meniscus, that is,
the free surface of the solution in which the molecules were first dissolved
 Fluorescent in situ Hybridization (FISH)
The detection of nucleotidic sequences on a
combed DNA molecule is peformed indirectly,
by first hybridizing the seeked nucleotidic
sequences (the probes) with the combed DNA
(also called the matrix DNA or target).
If the probes are synthesized with incorporated
fluorescent molecules or antigenic sites which
can be recognized with fluorescent antibodies,
the direct visualization of the relative position of
the probes is possible.
Green signals indicate positive hybridization of a YAC from human
3q26.3 to metaphase chromosomes from a patient with
Cornelia de Lange syndrome with a de novo balanced
translocation (breakpoints at 3q26.3 and 17q23.1). Red signals
indicate simultaneous hybridization with a chromosome 17
centromere probe.
 Long-range restriction mapping
Restriction mapping permits molecular mapping with
resolutions which depend on the frequency of the
recognition site.
Most restriction enzymes which recognize a 4 or 6 bp
sequence typically cut vertebrate DNA once every few
hundred or few thousand base pairs.
The recognition sequences for rare-cutter restriction
nucleases are typically 68 bp long and contain one or
more CpG dinucleotides which are rare in vertebrate
DNA
As a result, they generate fragments that are typically
several hundred kilobases in size.
 Pulse field electrophoresis

.
Description Gene Chromosome
Apolipoprotein E APOE 19
Ataxia telangiectasia ATM 11

.
Breast cancer, type 1
Breast cancer, type 2
BRCA1
BRCA2
17
13
Burkitt lymphoma MYC 8
Duchenne muscular dystrophy DMD X
Gaucher disease GBA 1
Juvenile onset diabetes IDDM1 6
Malignant melanoma CDKN2 9
Marfan syndrome FBN1 15
Neurofibromatosis, type 2 NF2 22
Obesity OBS 7
Phenylketonuria PAH 12
Polycystic kidney disease PKD1 16
Retinoblastoma, type 1 RB1 13
Severe combined immunodeficiency ADA 20
Small cell lung carcinoma SCLC1 3
 The BRCA1 gene maps to chromosome 17

.
.