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PV92 PCR/Informatics Kit

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Title: PV92 PCR/Informatics Kit


1
PV92 PCR/Informatics Kit
  • Population Genetics and Informatics

2
To estimate frequency of Alu within a
population
  • Amplify Alu insert from representative sample
    population
  • Calculate the expected allelic and genotypic
    frequencies
  • Perform Chi-squared Test

3
Calculating Allelic and Genotypic Frequencies
  • Within your class, how unique is your particular
    combination of Alu alleles? By calculating an
    allele frequency, you can begin to answer this
    question. An allele frequency is the percentage
    of a particular allele within a population of
    alleles. It is expressed as a decimal. You can
    calculate an allele frequency for the Alu PV92
    insertion in your class by combining all your
    data. For example, imagine that there are 38
    students in your class and the genotype
    distribution within the class is as follows

Genotype / /- -/-
Total (N) of people 25
5 8 38
4
Calculating Allelic Frequencies allele
  • Total number of alleles 2N 2(38) 76
  • Number of alleles
  • / 25 with two alleles 50
    alleles
  • /- 5 with one alleles 5
    alleles
  • Total 55 alleles
  • Frequency of number of alleles 55
    0.72
  • total alleles
    76
  • Calculation for the alleles would be similar,
    and the result would be .28
  • Notice ( allele) (- allele) 1.0

5
Calculating Genotypic Frequencies
  • How does the distribution of Alu genotypes in
    your class compare with the distribution in other
    populations? For this analysis, you need to
    calculate a genotype frequency, the percentage of
    individuals within a population having a
    particular genotype. Remember that the term
    allele refers to one of several different forms
    of a particular genetic site whereas the term
    genotype refers to the specific alleles that an
    organism carries. You can calculate the frequency
    of each genotype in your class by counting how
    many students have a particular genotype and
    dividing that number by the total number of
    students.
  • Given the ethnic makeup of your class, might you
    expect something different?
  • How can you estimate what the expected frequency
    should be?

6
Calculating Observed Genotypic Frequencies
  • Genotype / (p2) /- (2pq)
    -/- (q2) Total (N)
  • of people 25 5
    8 38
  • Observed 0.66 0.13
    0.21 1.00
  • frequency
  • Calculation
  • / genotypic frequency with genotype
  • total number of people (N)
  • 25/38
  • .66

7
Alu and Population Genetics
  • If within an infinitely large population no
    mutations are acquired, no genotypes are lost or
    gained, mating is random, and all genotypes are
    equally viable, then that population is said to
    be in Hardy-Weinberg equilibrium. In such
    populations, the allele frequencies will remain
    constant generation after generation. Genotype
    frequencies within this population can then be
    calculated from allele frequencies by using the
    equation
  • p2 2pq q2 1.0
  • p and q are the allele frequencies for two
    alternate forms of a genetic site. The genotype
    frequency of the homozygous condition is either
    p2 or q2 (depending on which allele you assign to
    p and which to q). The heterozygous genotype
    frequency is 2pq.

8
Alu and Population Genetics
  • Hardy-Weinberg Equilibrium

p2 2pq q2 1
p
q
pp
pq
p
/ p2 /- 2pq -/- q2
q
pq
qq
9
Using Hardy-Weinberg
  • Determine p2, 2pq, and q2 values
  • Expected genotypic frequencies
  • p 0.72 , so q 0.28 since p q 1
    p2 2pq q2 1
  • (0.72)2 2 (0.72)(0.28) (0.28)2
    1
  • 0.52 0.40 0.08 1
  • p2 0.52
  • 2pq 0.40
  • q2 0.08

10
Calculate Expected Number of Genotypes
  • Expected number of genotype
  • Genotypic frequency x population number (N)
  • Genotype Expected number
  • / 0.52 x 38 20
  • /- 0.40 x 38 15
  • -/- 0.08 x 38 3

11
Chi Squared Test
  • Chi-square, a statistical test used for comparing
    observed frequencies with expected frequencies.
    The larger the chi-square value, the greater is
    the difference between the observed and the
    expected values.
  • When using the Chi-square analysis, we test the
    null hypothesis that there is no difference
    between samples (observed and expected) and we
    assume that if there is any difference, then it
    arose simply by chance and is not real.
  • Our null hypothesis is that your class is in
    Hardy-Weinberg equilibrium. Whether or not we can
    accept the null hypothesis is given by a p-value.
  • If the calculated p-value is less than 0.05, the
    null hypothesis is disproved the population is
    not in Hardy-Weinberg equilibrium.
  • If the p-value is greater than 0.05, the
    population may be in Hardy-Weinberg equilibrium
    we can not prove that it is not in Hardy-Weinberg
    equilibrium.

12
Chi Squared Test
  • As an example, lets say that Chi-square analysis
    of some data gives a p-value of 0.17. This means
    that there is a 17 probability that the
    difference between the observed and the expected
    values is due to chance. It also means that there
    is an 83 (100 - 17 83) probability that the
    difference is not due to chance the difference
    is real.

13
Chi Squared Test
Observed Expected
(O-E)2
E / 25 20 1.25
/- 5 15 6.66 -/-
8 3 8.33
Total 17.12 X2
Critical Value (from statistics table)
5.9 17.12 is above 5.9 so the ratio is not
accepted.
14
Allele Server
  • Cold Spring Harbor Lab
  • DNA Learning Center

15
http// vector.cshl.org
Click on Resources
16
Click on Bioservers
Click on Bioservers
17
Enter the Allele Server
18
Allele Server
Click on Manage Groups
19
Select Type of Data
Select Group
20
Your Group
Scroll down to Your Group
21
Click on Add Group
Click on Add Group
22
Fill Out Form
23
Click on Edit Group
Click on Edit Group
24
Fill out Completely
25
Click on Individuals Tab
Click on Individuals Tab
26
Add Each Student With Information
  • Add as much info as you can
  • Genotype ( /, /-, -/- )
  • Gender
  • Personal Info

27
Click on Done
Done
28
Select Your Group
Select and then press OK
29
Click to Analyze
Then Click Here
Click Here
30
Then Look at the Terse and Verbose Tabs
31
Extensions
  • Is your class in Hardy-Weinberg Equilibrium?
  • Compare your group to other existing groups.
  • Form an explanation for the origination of Alu
    and how it spread throughout different
    populations.
  • Have students do manual calculations first and
    then compare to the computer generated version.

32
Plotting Alu PV92 on a World Map
  • The Alu element first appeared tens of millions
    of years ago and since that time, it has been
    increasing within our genome at the rate of about
    one copy every 100 years.
  • It is difficult to tell how Alu arose. It shows a
    striking similarity to a gene (called 7SL RNA)
    that performs a vital function in our metabolism.
    But Alu , it seems, has no function. It is
    self-serving and, like a parasite, takes
    advantage of us for its own replication without
    providing us any advantage to our own survival.
  • Most Alu elements are fixed they are found at
    the same chromosomal site in every person on the
    planet. Fixed Alu elements must have arose very
    early in our evolution, well before Homo sapiens
    appeared. When modern humans did arise some
    200,000 years ago, the vast majority of our Alu
    insertions came to us already intact in our DNA.
  • The Alu PV92 insertion, however, is not fixed.
    This insertion may or may not be present on one
    or both of a persons number 16 chromosomes.
    Since not everyone has the Alu PV92 element, it
    must have arisen after the initial human
    population began growing.
  • It is a widely held belief that modern humans
    originated in Africa and then disseminated across
    the planet. Did the Alu PV92 insert arise in
    Africa or on some other continent during our
    spread across the globe?
  • In the following exercise, you will plot the
    allele frequencies for various populations on a
    world map and make some determination as to where
    this Alu arose and how it might have spread
    across continents.

33
.18
.20
.12
.18
.86
.53
.30
.52
.09
.26
.35
.96
.15
34
Classroom How To
  • We have worksheets for calculating allelic and
    genotypic frequencies for your class
  • BABEC has the materials for the world population
    data
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