GENOME MAPPING Ms.ruchi yadav lecturer amity institute of
biotechnology amity university lucknow(up)
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GENOME MAPPING GENETIC MAPPING PHYSICAL MAPPING
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GENOME MAPPING 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). Physical mapping uses molecular
biology techniques to examine DNA molecules directly in order to
construct maps showing the positions of sequence features,
including genes.
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DNA MARKERS FOR GENETIC MAPPING 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).
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Restriction fragment length polymorphisms (RFLP)
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RFLP DETECTION
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Restriction fragment length polymorphisms (RFLPs)
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Pedigree based on RFLP analysis
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Linkage analysis shows that the disease gene D lies between
markers c and d.
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RFLP Distance between RFLP markers is also defined in
recombination units or cM.
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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) 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.
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RAPD (Random Amplified Polymorphic DNA)
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Simple sequence length polymorphisms (SSLPs), Unlike RFLPs,
SSLPs can be multi-allelic as each SSLP can have a number of
different length variants.
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VNTRs - Minisatellites
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Microsatellites: simple tandem repeats (STRs)
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Simple tandem repeats (STRs)
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STRs Advantages Easy to detect via PCR Lots of polymorphism
Co-dominant in nature Disadvantage Initial identification,DNA
sequence information necessary
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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 The percentage of
recombinant progeny produced in a cross is called the recombination
frequency, which is calculated as follows:
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Recombination Frequency
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Recombination fraction
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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.
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LOD Score Analysis The likelihood ratio as defined by :-
L(pedigree| = x) L(pedigree | = 0.50) where represents the
recombination fraction and where 0 x 0.49. L.R. = The LOD score (z)
is the log 10 (L.R.)
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Method to evaluate the statistical significance of results.
Maximum-likelihood analysis, which estimates the most likely value
of the recombination fraction as well as the odds in favour of
linkage versus nonlinkage. Given by Conditional probability L(data
1 ), which is the likelihood of obtaining the data if the genes are
linked and have a recombination fraction of . Likelihood of
obtaining one recombinant and seven nonrecombinants when the
recombination fraction is is proportional to 1 (1) 7, Where: is, by
definition, the probability of obtaining a recombinant, (I ) 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 , the
recombination fraction and x,the genetic distance (in morgans):
=1/2(1-e -2x ) or equivalently, X=-1/2ln(1-2)
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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
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LIMITATIONS 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.
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PHYSICAL MAPPING
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Physical mapping Actual physical distances Units in base-pairs
Contigs of large DNA fragments Large insert DNA libraries (BACs,
PACs, etc) Restriction fragment fingerprinting Minimum tiling set
to cover entire genome Correlation of genetic and physical maps
Genetic marker screening EST screening BAC-end sequencing FISH
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PHYSICAL MAPPING 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.
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The basic methodology for restriction mapping
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Restriction mapping partial restriction
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Physical maps 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.
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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
The position on the chromosome at which hybridization occurs
provides information about the map location of the DNA sequence
used as the probe DNA in the chromosome is made single stranded
(denatured). 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.
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Can distinguish chromosomes by painting using DNA hybridization
+ fluorescent probes during mitosis
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FISH
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FISH 16 DNA appears as a yellow band on chromosome16, thus
locating this particular simple sequence to one site in the
genome.
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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
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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
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Genetic and physical maps may differ in relative distances and
even in the position of genes on a chromosome.
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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.
