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Transcript
Biology 423 Research Paper:
Genetics behind cloning of a human gene:
1 - 2 page outlines due Nov. 6
Writing a scientific paper:
Choose a topic: Pick a genetic disease for which the responsible gene has
been cloned. You can find lists of these diseases at the following Yahoo site:
http://dir.yahoo.com/Health/Diseases_and_Conditions/Genetic_Disorders/
The OMIM database is also a useful place to get information to start.
Please do not choose Cystic Fibrosis or
Fibrodysplasia ossificans progressiva.
Collect papers:
Look for genetics studies, family studies
as well as physiology, biochemistry and cell biology
Make a title – active statements work well
eg. Grant’s disease is caused by a defective potassium pump
Make an outline:
Define a title for each section – even each paragraph.
Use active statements again.
eg. Grant’s disease is a genetic disease that affects breathing
The Grant’s disease mutation is on Chromosome 7
A mutation in a potassium pump is linked to Grant’s disease
(The two above will be the main sections)
Expression of wild type potassium pump reverts Grant’s disease
effects in cultured cells
Grant’s potassium pump transcripts are expressed in lung cells
Treatment of patients with potassium pump blockers has no
effect on progress of disease.
Microarray analysis suggests potential treatment.
These can be used as section titles or as topic sentences (see below)
Choose figures and make them.
You can copy figures from some of the papers you read.
If so, cite them properly eg (from Smith et al., 1987).
The figure with its figure legend should be comprehensible
without reading the text.
Write paragraphs
First sentence is a topic sentence
Last sentence is a concluding sentence
active voice
don’t try to sound academic
define your terms, (I am not a medical doctor)
if referring to a figure, define the figure but do not duplicate the figure legend.
Maintain the same tense, either past or present.
Make an abstract to put at the beginning of the paper
summarizing the experiments you will describe,
the results and the conclusions.
Citations: When you discuss published work, cite the paper.
Do the citation in the first sentence in which the study is mentioned.
Eg. Seven large families with a high incidence of cystic fibrosis were
surveyed for DNA markers linked to the disease (Smith et al., 1987).
References: at the end of the section: in alphabetical order
Smith J, Jones, P.A. and White, K. 1987 Family studies map cystic fibrosis
to Chromosome 7 Genetics 130: 147-156.
Use the journal “Cell” as an example of how to format the paper,
the citations and the references.
There are several nice reference managers available.
We use EndNote for making bibliographies and storing references.
Mapping genes by recombination frequency
Test cross to monitor recombination between different genes
Frequency of recombination is directly related to distance
between genes (loci) on chromosome
Three point cross
Drosophila, a model organism for genetics
Traits for our three point cross
Body color; yellow vs wild type
Bristles: forked vs straight (wild type)
Crossveins: crossveinless vs wild type
Fig. 5.12
Test cross
vg b pr / vg+ b+ r+ X vg b pr / vg b pr
Punnet square:
Male
Female
vg+b+pr+ vg b+pr+ vg+b pr+ vg b pr+ vg+b+ pr vg b+pr vg+b pr vg b pr
Vg b pr vg+b+pr+ vg b+pr+ vg+b pr+ vg b pr+ vg+b+ pr vg b+pr vg+b pr vg b pr
1:1:1:1:1:1:1:1 ratio of phenotypes if genes are not linked
If genes are linked, parental combinations of
alleles are overrepresented in progeny
Fig. 5.12
3 genes, which is in the middle?
Fig. 5.13
Calculate distance between pairs: vg to pr: add up all classes with a
recombination event between vg+ and pr or vg and pr+
252 + 241 + 13 + 9 = 525
Divide by total number of chromosomes scored:
525/4197 X 100 cM = 12.5
Calculate distance between pairs: pr to b: add up all classes with a
recombination event between pr+ and b or pr and b+
131 + 118 + 13 + 9 = 271
Divide by total number of chromosomes scored:
271/4197 X 100 cM = 6.4
The distance between vg and b is the sum of the distance
between vg-pr and pr-b
12.3 + 6.4 = 17.7
Fig. 5.12
Fig. 5.15
How do we map genes in humans?
Relative association of markers:
Allelic variants will co-segregate if the genes are
closely linked on a chromosome.
Map distances depend on frequency of recombination
To map a human genetic trait:
Look for association between mapped
markers and a trait of interest
Markers can be traits, proteins or DNA sequences
Anything that is polymorphic can be mapped
We can translate map position into
DNA sequence by determining the linkage
between DNA-based markers and traits.
Pedigree Analysis: symbols
Screen family members for
DNA markers linked to trait
Screen family members for
RFLP markers linked to trait
RFLP polymorphisms reveal genetic differences
2. Separate DNA fragments by
size on an agarose gel
3. Hybridize to single copy
radioactive probe- Southern Blot
1. Cut genomic DNA with
Restriction enzymes
HindIII polymorphism is closely linked to disease
N
D
D N
N
D
D
N
N
Marker G8 from a randomly chosen phage clone with
a 17.6 kb human DNA insert.
N
Test degree of linkage: odds of linkage
Data looks like M1 is linked to SF.
Mother has two M1 alleles.
Her chromosome is uninformative, like a test-cross.
Father has two different M alleles.
Recombination of his alleles can be seen in this pedigree.
In this family, there are 8 informative chromosomes.
1 has a recombination event.
therefore, a rough estimate of map distance is 1/8X 100 cM = 12.5%
Odds of Linkage is
(Probability gene and marker are linked at a certain map distance)
divided by (Probability they are unlinked).
Maximum likelihood odds of linkage; Change estimated linkage
distance (θ) to get the best LOD score for the data.
example
In our case: best odds of linkage value for this pedigree is L(.10) = 6.3
Log of L or LOD = 0.8
Maximum LOD score = Zmax = 0.8
LOD score is used to determine if two traits are linked
in human pedigrees
LOD nomenclature
• θ = recombinant fraction
• L(θ) = odds of linkage at 0 > θ < 0.5
• L(0.5) = odds of independent assortment
• Log[L(θ)/L(0.5)] = log-of-odds ratio = LOD = Z
• LOD scores > 3 indicate linkage
• LOD scores < -2 indicate non-linkage
Example linkage of marker and trait
θ
Z
θ
Σ(Z)
0.001
-6.0
0.05
28.2
0.01
-3.0
0.1
31.2
0.05
-1.1
0.15
30.4
0.1
-0.4
0.2
27.8
0.2
0.1
0.25
24.0
0.3
0.2
0.3
19.4
0.4
0.1
04
9.0
Zmax = maximum likelihood score (MLS)
To achieve significant LOD score:
Combine odds of linkage for many families:
p1(L)/p1(NL) x p2(L)/p2(NL) xp3(L)/p3(NL)
In practice we combine the log of odds:
LOD1 + LOD2 + LOD3.
Continue until LOD > 3.0 before linkage is accepted
Linkage distance is based on the linkage distance that
gives the maximum value for the data.
If genes and markers are unlinked, the p(L)/p(NL)
will be <1.0 in some families and the final LOD
score will be negative (<0).
Therefore, as you add more families the
LOD score will only increase if the data
of the majority of families supports linkage.
Summary
Genetic distance between two genes is measured by the
recombination frequency of alleles of those genes.
If genes are closely linked, alleles for those traits will not
become recombined as often as if the genes are
more distant on a chromosome.
Genome maps can be made using combinations of data from
relative recombination distances of many loci.
Human genetic linkage between two traits or two genes is
measured using Log scores.
Log scores greater than 3.0 (1/1000) chance of non-linkage are
the minimum values accepted as evidence of linkage
in family studies.