GENETICE

MAPPING
&
RECOMBINATION
FREQUENCY
GENOME MAPPING
• BIOCHEMICAL MARKERS
• GENETIC MAPPING
• PHYSICAL MAPPING
Biochemical markers for genetic
analysis of yeast
• ADE2 :Requires adenine ,Grows only when
adenine is present in the medium
• CAN1: Resistant to canavanine, Grows in the
presence of canavanine
• CUP1 :Resistant to copper ,Grows in the
presence of copper
• CYH1 :Resistant to cycloheximide, Grows in
the presence of cycloheximide
• LEU2 :Requires leucine ,Grows only when
leucine is present in the medium
• SUC2: Able to ferment sucrose, Grows if
sucrose is the only carbohydrate in the medium
• URA3: Requires uracil ,Grows only when uracil
is present in the medium
Biochemical markers in Human
• In human the biochemical phenotypes that can
be scored by blood typing.
• These include the standard blood groups such
as the ABO series and also the human
leukocyte antigens (the HLA system).
• A big advantage of these markers is that many
of the relevant genes have multiple alleles.
• For example, the gene called HLA-DRB1 has at
least 290 alleles and HLA-B has over 400.
• This is relevant because If all the family
members have the same allele for the gene
being studied then no useful information can be
obtained.
 Chromosome
bands

Cytogenetic map
Genes located
by FISH

1 Linkage mapping

Genetic
markers

2 Physical mapping

Overlapping
fragments

3 DNA sequencing
 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.
 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.

Characteristics of ideal molecular marker:

 Have high level of polymorphism
 Should be evenly distributed throughout the entire
genome
 Genetic differences should be clearly visible
 Preferably Co-dominant in nature
 Have linkage to distinct phenotypes
 Types of DNA Markers
Based on whether markers can discriminate between Homozygotes and
Heterozygotes Molecular markers may be categorized as :

CODOMINANT MARKERS DOMINANT MARKERS

Examples: Examples:
RFLP , SNPs , CAPS , SSR , RAPD , AFLP , SCAR , AP-
Allozymes PCR
Thomas Hunt Morgan's Drosophila
melanogaster genetic linkage map.
DNA Markers
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 tetranucleotides.

 Single nucleotide polymorphisms (SNPs).

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.
RAPD (Random Amplified
Polymorphic DNA)
Restriction fragment length polymorphisms
(RFLP)
 The DNA molecule on the left has a polymorphic
restriction site (marked with the asterisk) that is not
present in the molecule on the right.
 The RFLP is revealed after treatment with the restriction
enzyme because one of the molecules is cut into four
fragments whereas the other is cut into three fragments.
RFLP DETECTION
RFLP
 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 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.
Amplified Fragment Length
Polymorphism (AFLP)
Simple sequence length polymorphisms (SSLPs)
 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
 Advantages
◦ Easy to detect via PCR
◦ Lots of polymorphism
◦ Co-dominant in nature

 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.
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.
How do we estimate, from the offspring of a
single family, the likelihood that two gene pairs
are linked?

 Recombination frequency
 LOD score
 Haldane mapping function
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
Calculation of Recombination Frequency

 The percentage of recombinant progeny produced in a

cross is called the recombination frequency, which is
calculated as follows:
Recombination Frequency
 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.
 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.”
 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 genetic distances of loci and
recombination frequencies.
 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.
Integration of MAP
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.
PHYSICAL MAPPING
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
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.
The basic methodology for
restriction mapping
Restriction mapping
 partial restriction
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.
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.
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.
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
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
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
Genetic vs. Physical Distance
Genetic and physical maps may differ in relative
distances and even in the position of genes on a
chromosome.