Transcript goals - s3.amazonaws.com

Module 12
Human DNA Fingerprinting and
Population Genetics
p2 + 2pq + q2 = 1
GOALS
• Understand what an Alu sequence is and how it
can be used to understand population equilibrium.
• Understand the importance of the PCR and its use
in the “DNA fingerprint”.
• Understand the use of the Hardy-Weinberg
theorem.
• Use the Chi-square analysis and see whether our
data fits in Hardy-Weinberg theorem
Genes and DNA
• Gene is a piece of DNA that codes for a
particular protein.
• Only 5% of the total DNA is present as genes.
• Other 95% DNA is called as non-coding
DNA.
• Intron - In eukaryotes, the non-coding
sequence is within the genes are transcribed
into RNA but are not coded into the protein.
• Exon – the region of the transcribed RNA
coded for the protein.
RNA SPLICING
Genomic DNA
exon 1
Intron 1
Intron 2
Exon 2
Exon 3
transcription
Pre-mRNA
Exon 1
Intron 1
Intron 2
Exon 2
Exon 3
splicing
mRNA
Exon 1
Exon 2
Exon 3
translation
protein
Alu sequence
• The sequence of interest for this experiment is a
short repetitive sequence seen within an intron
sequence.
• This sequence is referred to as an Alu sequence
after a restriction enzyme site that is located within
this 300 base pair length of DNA.
5’
ALU
3’
INTRON
• Alu sequences occur in the human genome about
500,000 times.
Experiment outline
• In our experiment, PCR will be used to amplify a short
nucleotide sequence from human PV92 locus on
chromosome 16.
• The object is to create a personal DNA fingerprint that
shows the presence(+) or absence (-) of the “Alu” DNA
sequence on the paternal and maternal chromosomes.
• We will use primers for the Alu sequence that will
generate a 550 bp fragment if the Alu sequence is not
present and an 850 bp fragment if the Alu sequence is
present through the application of the polymerase chain
reaction.
PV92
genotype
Homozygous (+/+)
DNA size of
PCR products
850 bp
Homozygous (-/-)
550 bp
Heterozygous (+/-)
850 + 550
bp
Polymerase Chain Reaction
(PCR)
• Technique developed by Kary Mullis in 1983
(not very old!).
• Used in gene mapping, cloning, DNA
sequencing and gene detections.
• Powerful tool for the human genome mapping.
• Widely used in forensic science, therapeutic,
pharmaceutical and medical diagnostic
purposes.
Contd…
• PCR produce a large amount of DNA in a
test tube from a very small amount of
starting material.
•
e.g. A small amount of DNA can be
taken from drop of blood, a hair follicle or
cheek cells to generate millions of copies.
• A specific stretch of DNA can be amplified,
such as an Alu sequence.
PCR Steps (See your handout)
1. Denaturation step- sample is heated to 94°C and
at this temperature template DNA strands
separate.
2. Annealing step- temperature is dropped to 60°C.
primers will anneal to the template strands.
3. Extension step- sample is heated to 72 °C and
polymerase extends the primers to make a
complete copies of DNA template strands.
• All these steps are automated in the PCR machine
DNA polymerase used in PCR
• Thermally stable polymerase is needed.
Why?
• Taq polymerase is used. Stable even at 94°c.
• Obtained from thermophilic bacteria,
Thermus aquaticus.
PCR Requires
1.
2.
3.
4.
Reaction buffers
Four DNA bases (deoxynucleotides of A,G,T,C)
DNA polymerase
Two DNA primers (a small stretch of DNA that
will recognize a particular sequence in the target
DNA sequence)
5. Minute amounts of the desired template strands.
PCR movie
PCR MOVIE
http://www.biorad.com/LifeScie
nce/pdf/ExpPCR
kit.pdf
Contd…
• we will separate these fragments by gel
electrophoresis to observe the DNA
Fingerprint of each individual with respect to
this Alu sequence at the PV92 locus.
• Homozygous(+/+) for the presence of the Alu
repeat gives a 850 product, Homozygous(-/-)
for the absence gives a 550 product and a
heterozygous(+/-) gives two products of 850
and 550.
PV92
genotype
Homozygous (+/+)
DNA size of
PCR products
850 bp
Homozygous (-/-)
550 bp
Heterozygous (+/-)
850 + 550
bp
Gel Electrophoresis Separates Shorter DNA
Fragments from Longer DNA Fragments.
Gel for Alu marker
Hardy-Weinberg Theorem
• Once we determine the genotype distribution for
the class (that is count how many students are
++, +-, and --), we apply the Hardy-Weinberg
Theorem to this data set.
• The Hardy-Weinberg Theorem says that the
sexual shuffling of alleles due to meiosis and
random fertilization has no effect on the overall
genetic structure of a population over generations
unless acted upon by agents other than sexual
recombination. What are these agents? (see
handout)
Hardy-Weinberg (cont.)
• The Hardy-Weinberg equation allows us to
calculate the frequencies of alleles in a gene
pool if we know the frequencies of genotypes
(which we know from our experiment)
• Population geneticists use:
– The letter p to represent the frequency of one
allele
– The letter q to represent the frequency of the
other allele
•
p
+ q = 1
Hardy-Weinberg Equation
p2
+
Frequency
of AA
genotype
2pq
+
Frequency
of Aa + aA
genotype
q2
= 1
Frequency
of aa
genotype
One application of this is to calculate the percentage of
the human population that carries an allele for a particular
inherited disease. See example in Campbell textbook.
Hardy-Weinberg Equation
p2
+
Frequency
of homozygous
(+/+)
genotype
2pq
+
q2
= 1
Frequency
Frequency
of heterozygous of homozygous
genotype
(-/-)
genotype
Chi-Square Analysis
• Lastly, we can make sure that our class is in
Hardy-Weinberg equilibrium by comparing
our observed genotypic data in class to
expected genotypic data
– Observed data: class genotypes
– Expected data: See website for
www.dnalc.org. May be provided on lab
report.
On 04/17,18……
• Isolation of cheek cell DNA .
• PCR reaction (to be set up by the TA’s)
• Practice gel electrophoresis.
Next Class (on 04/22,23..)
• Gel electrophoresis of the amplified cheek
cell DNA (done by TA’s)
• Calculate Hardy-Weinberg Equilibrium
equation.
• Calculate the Chi-square value.