Download Lab 12

DNA Paternity Test
RFLP analysis
(Restriction Fragment Length Polymorphism)
-analyze the size of DNA fragments that result
when a segment of DNA from the genome
is cut with special enzymes
-Restriction Enzymes cut DNA at specific
sequences
-each enzyme recognizes and cuts DNA at a
different base sequence
e.g. BamHI
XXXXXXXXGGATCCXXXXXXXXXX
XXXXXXXXCCTAGGXXXXXXXXXX
-due to spontaneous mutations over time,
different people have slightly different base
sequences in their DNA
-if mutation creates or deletes a restriction site
in the DNA, the new DNA will generate
more or less fragments/different sized
fragments when cut with a particular
enzyme
1
2
3
...XXGGATCCXXXXXXGGATCCXXXX...
...XXCCTAGGXXXXXXCCTAGGXXXX…
cut with BamHI generates three fragments
1
2
...XXGGATCCXXXXXXAGATCCXXXX...
...XXCCTAGGXXXXXXTCTAGGXXXX…
single base mutation, BamHI will not cut 2nd
site, generates only two fragments
-RFLP analysis targets DNA segments known
to have variability between individuals and
cuts with enzymes that show those
differences
-cut DNA fragments are then analyzed on
agarose gel electrophoresis
DNA Gel Electrophoresis
-use to separate DNA by size to visualize it
-Agarose gel = matrix with pores
-place in running chamber with electrolyte
buffer
-electrical current runs through buffer between
electrodes on opposite sides of gel
-DNA samples loaded into wells near
negative electrode
-DNA has negative charge due to phosphate
backbone
-DNA moves through gel away from negative
toward positive electrode
-gel matrix separates moving DNA by size:
-smaller molecules “squeeze” through gel
easier thus moving faster
-smaller molecules end up further away
from the wells
-DNA will need to be stained to see it after
running the gel
_
Bigger
+
Smaller
-each person has two copies of every gene:
one from their mother, one from their
father
-a resulting child’s DNA will have
characteristics of both parents’ DNA
Mom
200bp
500bp
800bp
1000bp
200bp
500bp
800bp
1000bp
Dad
200bp
1300bp
1000bp
200bp
1300bp
1000bp
Child
200bp
200bp
500bp
800bp
1300bp
1000bp
1000bp
(one gene from mom, one gene from dad)
Dad
Child
Mom
_
1300bp
1000bp
800bp
500bp
+
*do practice problem on worksheet
200bp
8. Given the following DNA agarose gel electrophoresis RFLP analysis, who is the father
of the child in question?
Mother
Child
Dad 1
Dad 2
Dad 3
8. Given the following DNA agarose gel electrophoresis RFLP analysis, who is the father
of the child in question?
Mother
Child
Dad 1
Dad 2
Dad 3
First circle and ignore all the fragments that
could have been inherited from the mom...
8. Given the following DNA agarose gel electrophoresis RFLP analysis, who is the father
of the child in question?
Mother
Child
Dad 1
Dad 2
Dad 3
Then find the only man who could have
contributed all the remaining fragments to the
child
Agarose gel electrophoresis = “submarine gel”
-submerged in running buffer
-DNA must be suspended in loading buffer:
contains:
1. glycerol to make sample dense to
sink through running buffer into well
2. tracking dye to follow movement
through gel (DNA is colorless)
-bromophenol blue: co-migrates with
~300bp (small DNA)
-xylene cyanol: co-migrates with
~4000bp (big DNA)
-After gel runs, DNA must be stained with
methylene blue to visualize it
*practice use of pipettors
*practice gel loading
*set up and run pre-cut paternity test DNA
samples
Genetics and Inheritance
Genetic diversity comes from:
1. Meiotic recombination
2. Random fertilization
Play GeneticVariation.swf
Human Genetics and Inheritance
Humans: 46 chromosomes
23 homologus pairs
(one of each from mom, one of each from dad)
-2 copies of each gene, each is 1 allele
-allele = a version of a gene
-the 2 alleles could be the same or they could
be different:
same = homozygous (GG or gg)
different = heterozygous (Gg)
genotype = ones genetic makeup: all the
alleles a person has
phenotype = appearance of a person due to the
genes/alleles
-many traits or phenotypes will depend on the
contribution of many alleles
-some traits are determined by a single pair of
alleles
-in this case, one allele type will be
dominant over another and the
dominant allele will be the phenotype
e.g.
alleles G and g
G = green spots (dominant, capital letter)
g = no spots (recessive, lower case letter)
Genotype GG or Gg = Phenotype green spots
Genotype gg = Phenotype no spots
In order to have the recessive phenotype, both
alleles have to be recessive
A single dominant allele will control the
phenotype
*do genetics problems in book
*do genetics problems on worksheet
Note for genetics problems:
Phenotype = what it looks like e.g. freckles
Genotype = the genes: write as letters, but say:
- “homozygous dominant” (FF)
- “homozygous recessive” (ff)
- “heterozygous” (Ff)