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BIOL 205 Mendelian and Molecular
Genetics
Lecture : 11
Three point testcrossDeducing gene order
Interference
Using ratios as diagnosticsMapping with molecular makers
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Throw knives at this chromosome!
The larger the distance between two loci the morechance of recombination
Recombination frequencies less than 50% indicate
linkage. Remember unlinked genes on different
chromosomes show 50% recombinant (due toindependent assortment). So as we approach 50%
recombination, genes that are linked but far from each
other will appear to be unlinked.
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Figure 4-10
Longer regions have more crossovers and thus higher
recombinant frequen
cies
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Three Point test cross
v= vermillion eyes
cv= crossveingless
ct= cut wing edges
P v+/v+. cv/cv. ct/ct X v/v. cv+/cv+. ct+/ct+
Q. Are they linked and if so
what order on thechromosome
Gametes: v+. cv. ct v. cv+. ct+
F1 trihybrid: v+/v. cv/cv+. ct/ct+
(inputs)
(order or linkage not known)
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Trihybrid females are test crossed with triple recessive males:
v+/v. cv/cv+. ct/ct+ v/v. cv/cv. ct/ctX
F1 Trihybrid female Tester male*
How many gametes from trihybrid?
23= 2X2X2= 8
*Test cross is usually done with tester male because
Drosophila males do not show meiotic recombination.
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How many gametes from trihybrid? Use branch diagram
v cvcv+
ct
ct+
ct
ct+
v. cv. ct
v. cv. ct+
v. cv+. ct
v. cv+. ct+
v+ cvcv+
ct
ct+
ct
ct+
v
+
.
cv
.
ct
v+. cv. ct+
v+. cv+. ct
v+. cv+. ct+
Parental
Parental
Recombinants
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Trihybrid females are test crossed with triple
recessive males:
v+/v. cv/cv+. ct/ct+ v/v. cv/cv. ct/ctX
F1 Trihybrid female Tester male
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Gametes
v+. cv. ctv. cv+. ct+
v. cv. ct+
v+. cv+. ct
v. cv. ct
v+. cv+. ct+
v. cv+. ct
v+. cv. ct+
580
592
45
40
89
94
3
5
cvand ct
Recombinant for loci
R
R
1448
vand cv
R
R
R
R
268
(18.5%)
vand ct
R
R
RR
191
(13.2%)
RR
93
(6.4%)
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v ct cv
13.2 m.u. 6.4 m.u.
Q. Why doesnt the vand cvRF add up to 19.6%?
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Figure 4-11
When we calculated the RF value forvand cvwe did not
count the v ct cv+ and v+ct+cvgenotypes; after all, with
regard to vand cv, they are parental combinations (v cv+
and v+cv). This leads to an underestimation of the
recombinant frequency.
Normally this not a problem as the sum of the two shorter
distances gives us the best estimate of overall distance
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v ct cv
13.2 m.u. 6.4 m.u.
Recombination between v and cv :
45+40+89+94+3+3+5+5= 284/1448= 0.196
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Are the crossovers in adjacent chromosome
regions independent events or does a crossover in
one region affect the likelihood of there being a
crossover in an adjacent region?
Generally, crossovers inhibit each other somewhat
in an interaction called interference.
In some regions, there are never any observed
double recombinants (complete interference).
Interference values anywhere between between 0(no interference) and 1 (complete) and are found in
different regions of the chromosome and in
different organisms.
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A recombination-based map of one of the chromosomes ofDrosophila
cv ct 20 -13.7= 6.3m.u.
v ct 33 - 20= 13 m.u.
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Deducing gene order by inspectionGene order can usually be deduced by inspection,
without a recombinant frequency analysis. Typically, forthree linked genes, we have the eight genotypes at the
following frequencies:
two at high frequency
two at intermediate frequency
two at a different intermediate frequency
two rare
(Parental)(Recombinants)
(Recombinants from double crossovers)With three genes only three gene orders are possible, each
with a different gene in the middle position.
The gene in the middle is that it is the allele pair that has
flipped position in the double-recombinant classes.
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Different gene orders give different double recombinants
v ct cv
13.2 m.u. 6.4 m.u.
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Figure 4-13c
A map of the 12 tomato chromosomes
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Mapping with Molecular Markers
Changes in the DNA
Eg. Single nucleotide polymorphism (SNPs) GC to ATEg. DNA repeats eg. Microsatellite DNA repeats
In humans, there are thought to be about 3 million SNPs
distributed more or less randomly at a frequency of 1 in every
300 to 1000 bases, providing a useful set of markers for fine-
scale mapping.
Where are they?
They can affect a gene leading to a phenotype. Eg.
PKU or Albinism. You can be either homozygous orheterozygous for a SNP.
But SNPs do not have to be in a gene or affect a
gene.
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Why do we use molecular markers?
1)They have a position on the the chromosome2)We can detect them by sequencing, RFLP or
PCR
3)The phenotype is the actual behavior of theDNA on Gel electrophoresis4)Molecular markers can be used as a pseudo test
cross because we can determine the
homozygous, or heterozygous states of eachmorph.
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How do we detect SNPs?
If you know the sequence in the region of theSNPyou can just sequence.
RFLPs: restriction fragment length
polymorphisms (RFLPs), which areSNPs located at a restriction enzyme's
target site.
They do not have to be in a gene, butwe can use them to ask is our gene is
linked to a RFLP?
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Figure 4-15a
An RFLP linked to a mouse disease gene
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Figure 4-15b
An RFLP linked to a mouse disease gene
Q. Is D linked to M1 or M2?
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Figure 4-15c
An RFLP linked to a mouse disease gene
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Minisatellite and microsatellite markersmost genomes contain a great deal of repetitive DNA. called
simple sequence length polymorphisms (SSLPs). They arealso sometimes called variable number tandem repeats orVNTRs.
SSLPs commonly have multiple versions; sometimes 4
versions (2 from each parent) can be tracked in a pedigree.
Two types of SSLPs are useful in mapping and other genomeanalysis: minisatellite and microsatellite markers.minisatellite marker: is based on variation in the number of
tandem repeats of a repeating unit from 15 to 100 nucleotides
long. In humans, the total length of the unit is from 1 to 5 kb.
Detected by Southern Blot.microsatellite marker: is based on variable numbers oftandem repeats of an even simpler sequence, generally a
dinucleotide. Detected by PCR.
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The most common microsatellite type is a repeat
of CA and its complement GT, as in the following
example:
5 C-A-C-A-C-A-C-A-C-A-C-A-C-A-C-A 3
3 G-T-G-T-G-T-G-T-G-T-G-T-G-T-G-T 5
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Figure 4-19a
A microsatellite locus can show linkage to a disease gene
P
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P/p . M/M p/p. M/M
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Figure 4-20
Phenotypic and
molecular markers
mapped on human
chromosome 1
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