Transcript Genetic_mapping-100917050507

GENOME MAPPING
Ms.ruchi yadav
lecturer
amity institute of biotechnology
amity university
lucknow(up)
GENOME MAPPING
GENETIC MAPPING
PHYSICAL MAPPING
GENOME MAPPING
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Genetic mapping is based on the use of
genetic techniques to construct maps
showing the positions of genes and other
sequence features on a genome.
◦ Genetic techniques include cross-breeding
experiments or,
◦ Case of humans, the examination of family
histories (pedigrees).
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Physical mapping uses molecular biology
techniques to examine DNA molecules
directly in order to construct maps showing
the positions of sequence features, including
genes.
DNA MARKERS FOR GENETIC MAPPING
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Mapped features that are not genes are called DNA
markers. As with gene markers, a DNA marker must have
at least two alleles to be useful. There are three types of
DNA sequence feature that satisfy this requirement:
Restriction fragment length polymorphisms (RFLPs)
Simple sequence length polymorphisms (SSLPs), and
i) Minisatellites, also known as variable number of tandem
repeats (VNTRs) in which the repeat unit is up to 25
bp in length;
ii) Microsatellites or simple tandem repeats (STRs),
whose repeats are shorter, usually dinucleotide or
tetranucleotide units.
single nucleotide polymorphisms (SNPs).
Restriction fragment length polymorphisms
(RFLP)
RFLP DETECTION
Restriction fragment length polymorphisms
(RFLPs)
Pedigree based on RFLP analysis
Linkage analysis shows that the disease gene D lies between
markers c and d.
RFLP
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Distance between RFLP markers is also
defined in recombination units or cM.
Amplified Fragment Length Polymorphism
(AFLP)
AFLPs are differences in restriction
fragment lengths caused by SNPs or
INDELs that create or abolish restriction
endonuclease recognition sites.
 The AFLP technique is based on the
selective PCR amplification of restriction
fragments from a total digest of genomic
DNA
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RAPD (Random Amplified Polymorphic
DNA)
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RAPD markers are DNA fragments from PCR
amplification of random segments of genomic
DNA with single primer of arbitrary
nucleotide sequence.
RAPD does not require any specific
knowledge of the DNA sequence of the
target organism
The identical 10-mer primers will or will not
amplify a segment of DNA, depending on
positions that are complementary to the
primers' sequence.
RAPD (Random Amplified Polymorphic
DNA)
Simple sequence length polymorphisms (SSLPs),
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Unlike RFLPs, SSLPs can be multi-allelic as each SSLP can
have a number of different length variants.
VNTRs - Minisatellites
VNTRs - Minisatellites
Microsatellites: simple tandem
repeats (STRs)
Simple tandem repeats (STRs)
STRs
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Advantages
◦ Easy to detect via PCR
◦ Lots of polymorphism
◦ Co-dominant in nature
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Disadvantage
◦ Initial identification,DNA sequence
information necessary
MAPPING TECHNIQUES
Linkage analysis is the basis of genetic mapping.
 The offspring usually co-inherit either A with B or a
with b, and, in this case, the law of independent
assortment is not valid.
 Thus to test for linkage between the genes for two
traits, certain types of matings are examined and
observe whether or not the pattern of the
combinations of traits exhibited by the offspring
follows the law of independent assortment.
 If not, the gene pairs for those traits must be linked,
that is they must be on the same chromosome pair.
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What types of matings can reveal that the
genes for two traits are linked?
Only matings involving an individual who is
heterozygous for both traits (genotype AaBb) reveal
deviations from independent assortment and thus reveal
linkage.
 Moreover, the most obvious deviations occur in the
test cross, a mating between a double heterozygote
and a doubly recessive homozygote (genotype aabb).
 Individuals with the genotype AaBb manifest both
dominant phenotypes; those with the genotype aabb
manifest both recessive phenotypes.
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How do we estimate, from the offspring of a
single family, the likelihood that two gene pairs
are linked?
Recombination fraction
 LOD score
 Haldane mapping function
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Recombination Frequency
Recombination fraction is a measure of the distance
between two loci.
 Two loci that show 1% recombination are defined as
being 1 centimorgan (cM) apart on a genetic map.
 1 map unit = 1 cM (centimorgan)
 Two genes that undergo independent assortment have
recombination frequency of 50 percent and are located
on nonhomologous chromosomes or far apart on the
same chromosome = unlinked
 Genes with recombination frequencies less than 50
percent are on the same chromosome = linked
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Calculation of Recombination Frequency
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The percentage of recombinant progeny produced in a
cross is called the recombination frequency, which is
calculated as follows:
Recombination Frequency
Recombination fraction
LOD SCORE
• The LOD score is calculated as follows:
• LOD = Z = Log10 probability of birth sequence with a given linkage
probability of birth sequence with no linkage
• By convention, a LOD score greater than 3.0 is
considered evidence for linkage.
• On the other hand, a LOD score less than -2.0 is
considered evidence to exclude linkage.
LOD Score Analysis
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The likelihood ratio as defined by :L(pedigree| = x)
L(pedigree |  = 0.50)
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where  represents the recombination
fraction and where 0  x  0.49.
( (1   ) )
 L.R. =
N
(  0.5)
R
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NR
The LOD score (z) is the log10 (L.R.)
Method to evaluate the statistical significance of
results.
Maximum-likelihood analysis, which estimates the “most
likely” value of the recombination fraction Ø as well as
the odds in favour of linkage versus nonlinkage.
 Given by Conditional probability L(data 1 Ø),
which is the likelihood of obtaining the data if the genes
are linked and have a recombination fraction of Ø.
 Likelihood of obtaining one recombinant and seven
nonrecombinants when the recombination fraction is Ø is
proportional to Ø1(1–Ø)7,
Where: Ø is, by definition, the probability of obtaining a
recombinant ,
(I – Ø) is the probability of obtaining a nonrecombinant.
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Mapping function
The genetic distance between locus A and locus B is
defined as the average number of crossovers
occurring in the interval AB.
 Mapping function is use to translate recombination
fractions into genetic distances.
 In 1919 the British geneticist J, B. S. Haldane
proposed such Mapping function
 Haldane defined the genetic distance, x, between
two loci as the average number of crossovers per
meiosis in the interval between the two loci.
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What is Haldane ’s mapping function ?
Assumptions: crossovers occurred at random along
the chromosome and that the probability of a
crossover at one position along the chromosome
was independent of the probability of a crossover at
another position.
 Using these assumptions, he derived the following
relationship between
 Ø, the recombination fraction and
 x ,the genetic distance (in morgans):
Ø=1/2(1-e-2x) or equivalently,
X=-1/2ln(1-2Ø)
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Genetic distance between two loci increases, the
recombination fraction approaches a limiting value
of 0.5.
 Cytological observations of meiosis indicate that
the average number of crossovers undergone by
the chromosome pairs of a germ-line cell during
meiosis is 33.
 Therefore, the average genetic length of a human
chromosome is about 1.4 morgans, or about 140
centimorgans.
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Integration of MAP
LIMITATIONS
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A map generated by genetic techniques is rarely sufficient
for directing the sequencing phase of a genome project.
This is for two reasons:
The resolution of a genetic map depends on the number
of crossovers that have been scored .
◦ Genes that are several tens of kb apart may appear at
the same position on the genetic map.
Genetic maps have limited accuracy .
◦ Presence of recombination hotspots means that
crossovers are more likely to occur at some points
rather than at others.
◦ physical mapping techniques has been developed to
address this problem.
PHYSICAL MAPPING
Physical mapping
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Actual physical distances
◦ Units in base-pairs
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Contigs of large DNA fragments
◦ Large insert DNA libraries (BACs, PACs, etc)
◦ Restriction fragment fingerprinting
◦ Minimum tiling set to cover entire genome
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Correlation of genetic and physical maps
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Genetic marker screening
EST screening
BAC-end sequencing
FISH
PHYSICAL MAPPING
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Restriction mapping, which locates the
relative positions on a DNA molecule of the
recognition sequences for restriction
endonucleases;
Fluorescent in situ hybridization (FISH), in
which marker locations are mapped by
hybridizing a probe containing the marker to
intact chromosomes;
Sequence tagged site (STS) mapping, in which
the positions of short sequences are mapped
by PCR and/or hybridization analysis of
genome fragments.
The basic methodology for
restriction mapping
Restriction mapping
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partial restriction
Physical maps
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Physical maps can be generated by aligning the
restriction maps of specific pieces of cloned genomic
DNA (for instance, in YAC or BAC vectors) along the
chromosomes.
These maps are extremely useful for the purpose of
map-based gene cloning.
Fluorescent in situ hybridization
(FISH)
FISH enables the position of a marker on a
chromosome or extended DNA molecule
to be directly visualized
 In FISH, the marker is a DNA sequence
that is visualized by hybridization with a
fluorescent probe.
 In situ hybridization intact chromosome is
examined by probing it with a labeled DNA
molecule.
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In situ hybridization with radioactive
or fluorescent probes
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The position on the chromosome at which
hybridization occurs provides information about the
map location of the DNA sequence used as the probe
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DNA in the chromosome is made single stranded
(‘denatured’).
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The standard method for denaturing chromosomal
DNA without destroying the morphology of the
chromosome is to dry the preparation onto a glass
microscope slide and then treat with formamide.
Can distinguish chromosomes by “painting” –
using DNA hybridization + fluorescent probes –
during mitosis
FISH
FISH
16
16
DNA appears as a yellow band on chromosome16, thus locating this
particular simple sequence to one site in the genome.
Sequence tagged site (STS) mapping
A sequence tagged site or STS is simply a
short DNA sequence, generally between
100 and 500 bp in length, that is easily
recognizable and occurs only once in the
chromosome or genome being studied.
 To map a set of STSs, a collection of
overlapping DNA fragments from a single
chromosome or from the entire genome
is needed
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STS mapping
STS mapping
The data from which the map will be derived
are obtained by determining which
fragments contain which STSs.
 The chances of two STSs being present on
the same fragment will, of course, depend on
how close together they are in the genome.
 The data can therefore be used to calculate
the distance between two markers
 Each map distance is based on the frequency
at which breaks occur between two markers
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Genetic vs. Physical Distance
Map distances based on recombination
frequencies are not a direct measurement
of physical distance along a chromosome
 Recombination “hot spots” overestimate
physical length
 Low rates in heterochromatin and
centromeres underestimate actual
physical length
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Genetic vs. Physical Distance
Genetic and physical maps may differ in relative
distances and even in the position of genes on a
chromosome.
Map-based sequencing
Map-based sequencing The first method
for assembling short, sequenced
fragments into a whole-genome sequence,
called a map-based approach,
 Requires the initial creation of detailed
genetic and physical maps of the genome,
 It provide known locations of genetic
markers (restriction sites, other genes, or
known DNA sequences) at regularly
spaced intervals along each chromosome.
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Map-based sequencing