Genetic and Physical Mapping
Lecture 2
Why map genes?
1. Many diseases are partially genetic
– Also: environmental factors, randomness
2. We want to identify these genes
– Early diagnosis for abortion or regular checks
– First step towards developing treatment
3. Individual sequencing is too costly (today)
– Sequence a small number of markers
– Analyze statistically via biological principles
Genetic mapping:
based on the use of genetic techniques to construct
linkage maps showing the positions of genes and other
sequence features in a genome
Physical mapping:
uses molecular biology techniques to examine DNA
molecules directly in order to construct maps showing
the exact position of genes and other sequences
Genetic Linkage Maps
1. A genetic linkage map shows the relative locations of specific DNA
markers along the chromosome. Any inherited physical or molecular
characteristic that differs among individuals and is easily detectable in
the laboratory is a potential genetic marker
2. Markers can be expressed DNA regions (genes) or DNA segments that
have no known coding function but whose inheritance pattern can be
followed. DNA sequence differences are especially useful markers
because they are plentiful and easy to characterize precisely
3. Markers must be polymorphic to be useful in mapping; that
is, alternative forms must exist among individuals so that they are
detectable among different members in family studies
4. Polymorphisms are variations in DNA sequence that occur on average
once every 300 to 500 bp. Variations within exon sequences can lead
to observable changes, such as differences in eye color, blood
type, and disease susceptibility
Stages of Mapping a Gene
• Demonstrate disease is hereditary
– Show it runs in families
• Linkage analysis to identify region
– Widely-spaced markers, e.g. RFLPs
• Association analysis to narrow region
– Closely-spaced markers, usually SNPs
• Clone the gene within found region
– Investigate its metabolic relevance
Summary
1. A genetic linkage map shows the relative locations of
specific DNA markers along the chromosome
2. Five major DNA markers for genetic mapping:
RFLP
RAPD
SNP
AFLP
Microsatellites
3. A physical map shows the exact position of genes and
other sequences on the chromosome
4. Five physical mapping methods:
optical mapping
fingerprinting
chromosome walking,
STS and FISH
Types of DNA Markers
1. Restriction fragment length polymorphisms (RFLP)
2. RAPD-Random amplified polymorphic DNA (RAPD)
3. Microsatellite, simple sequence repeat (SSR)
markers or short tandem repeat (STR)
4. Single nucleotide polymorphisms (SNPs)
5. Amplified fragment length polymorphism (AFLP)
Restriction Fragment Length Polymorhisms =
RFLP
Minisatellites
Variable number of tandem repeats = VNTR
High number of alleles
High level of heterozygocity
Problem: southern blots and radioactive
probes, minisatellites are to long to be amplified
by PCR
Not evenly spread over the genome
Microsatellites
Single Nucleotide Polymorphisms (SNP)
Restriction Fragment Length Polymorhisms = RFLP
Minisatellites
Microsatellites
Standard tools for mapping studies and linkega
analysis
Mostly (CA)n repeats
Tri-en tetranucleotide repeats replace more and more
the dinucleotide repeats, because of the better results
Dinucleotide-repeats are volnurable to “replication
slippage” during PCR as such that each allel can give a
small ladder on a gel
Development of multiplex PCR reactions
Single Nucleotide Polymorphisms (SNP)
Physical Mapping
Construction of a physical map which consists of
continuous overlapping fragments of cloned DNA that has the
same linear order as found on the chromosomes from which
they were derived
Physical Map vs Genetic Map Limitations of
genetic maps
1. The resolution of a
genetic map depends on the
number of crossovers that
have been scored. Limited
resolution (1cM is approx. 1
Mb)
2. Genetic maps have
limited accuracy. Certain
genomic regions more
sensitive to recombination
3. Markers must be
polymorphic for genetic
mapping
Physical Map of a Chromosome
Contig:
A series of overlapping clones or sequences that
collectively span a particular chromosomal region
Depicts genetic markers and DNA sequences between the
markers measured in base pairs (High Resolution)
Physical Mapping Methods
1. Optical mapping
2. Restriction fragment fingerprinting
3. Chromosome walking
4. Sequence tagged site (STS) mapping
5. Fluorescent in situ hybridization (FISH)
2 Restriction fragment fingerprinting
Individual clones are digested with different
restriction enzymes
The digested DNA is labeled with radioactive or
fluorescent dye and run on a sequencing gel
The fingerprint data is collected and analyzed
for contig assembly
Disadvantages: labor intensive and difficult to fill
gaps.
3 Chromosome walking
Markers with known map position are used as
probe to screen the large insert library
Clones hybridizing with the same single copy
marker are considered to be overlapping
PCR amplification of DNA pools using primers
derived from DNA markers with known position was
also used for physical map construction
Disadvantage:
Labor intensive, repetitive sequence
misleading, markers unevenly distributed in the genome.
Chromosome Walking
4 Sequence Tagged Sites (STSs)
An STS is a short region of DNA about 200-300
bases long whose exact sequence is found nowhere
else in the genome
Two or more clones containing the same STS must
overlap and the overlap must include the STS
Disadvantages:
still very labor intensive and
high expensive for primer synthesis
STS mapping of linked markers and BAC clones
PCR confirmation of STS markers in the genome Each
STS contains a unique sequence
5 Fluorescence in situ hybridization (FISH)
This technique uses synthetic polynucleotide strands or
short DNA fragment that bear sequences known to be
complementary to specific target sequences at specific
chromosomal locations.
The polynucleotides are bound via a series of link
molecules to a fluorescent dye that can be detected by a
fluorescence microscope
•Human metaphase chromosomes hybridized to fluorescent probes from
two overlapping micro-dissection libraries.
•Probes specific to chromosome regions 1p34– 35 and 1p36 were labeled
using the ULYSIS Oregon Green 488 (U21659) and Alexa Fluor 594
(U21654) Nucleic Acid Labeling Kits, respectively.
(http://www.probes.com/servlets/photohigh?fileid=g001276)
FISH mapping of BACs (bacterial artificial chromosomes)
Application of FISH in physical mapping
Jackson S. et al. 2000. Genetics, 156: 833-838
Combination of fingerprinting, molecular linkage
map, STS, end sequencing and FISH mapping
Combination of STS markers, BACs and FISH
Human genome anatomy:
BACs integrating the genetic and cytogenetic maps
for bridging genome and biomedicine. Genome Res.
1999 9(10):994-1001
872 unique STSs and 957 BACs were used
Gene mapping applications.
1. Useful for locating the position of genes on
chromosomes, e.g. if two genes are closely linked and the
position of one is known, then the other must also be
nearby.
2. Useful in estimating genetic risk, e.g. if a gene cannot be
tested directly, then variation at a closely linked locus may
indicate the presence or absence of a detrimental allele.
3. Has aided in:
Mapping of all human genes (Human Genome Project)
Mice,
Drosophila,
Caenorabditis elegans , Arabidopsis thaliana
Yeast, Escherichia coli.
By 1999:
Yeast; E. coli; C. elegans
A dozen other bacteria have been completely
sequenced and all their genes identified, although the
functions of most are unknown.
Major progress has been made in mapping human
genes, and a "rough draft" of the human genome was
read by 2000.
Understanding of function of the many newly discovered
human genes is being greatly aided by the studies of
yeast, which has many genes similar to those of humans.
Resources
• Genetic Analysis Software
– http://linkage.rockefeller.edu/soft/list.html
• Introduction to Genetic Analysis
– http://www2.qimr.edu.au/davidD/Course/
• Genetic Analysis Resources
http://ihome.cuhk.edu.hk/~b400559/complexdismap.html
• NCBI Human Genes and Disease
– http://www.ncbi.nlm.nih.gov/disease/
• International Haplotype Map Project
– http://www.genome.gov/10001688
