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					                          Alu stuff to learn about on April 8, 2010

   By the end of the period students should be able to:
        Know where they can go to look up factoids about Hardy-Weinberg equilibrium
        Be developing an understanding of HWE
        Know what allele frequencies are
        Know how to calculate allele frequencies
        Be familiar enough with the terms + allele and – allele and the controls used in
          the Alu lab to know the differences between the positive and negative terms
        Evaluate the world map of p allele frequencies so they can hypothesize what
          their genotype is- to be finished for homework if not completed in class

Hardy Weinberg Equilibrium (HWE)
    Quick facts and examples on curriculum card (written by Ms Getz)
    The textbook

Allele frequencies and Alu
     Alleles are different versions of a specific region of DNA
           o For something like Alu, what we are looking at are segments of
              chromosomes that have been changed because they have a 300 bp insert in a
              specific region that was not there previously
           o The assumption is that the Alu segments/fragments were not a part of the
              original organism that evolved. They are segments of DNA that find a new
              location at some point in time. This point in time could have been a few
              hundred thousand years ago or a million years ago.
           o The relocation of Alu has to have happened in germ line cells- the ones that
              give rise to egg and sperm so that they could be passed on to future
           o The Alu segment we’re investigating is not in a gene. It is in a location that at
              this point in time we know of no use for this segment of DNA other than it
              being a region of space on chromosome 16. Yes there are Alu segments that
              have landed in the middle of genes and these interruptions of the gene have
              led to human diseases, but the mutation we are looking at has no known
              significance. We would not be looking at a DNA sequence that has any
              significant genetic information because this is not the venue for you to learn
              about a meaningful mutation you may have.
           o Alu mutations show up in other hominids
           o If you research Alu elements, the one we are looking at is Pv92. Pv92, being
              innocuous, is used in research to track evolutionary migratory paths. It can
              also be used as part of parentage tests, but it is not one of the regions looked
              at for CODIS analysis. CODIS looks at regions of DNA where there are more
              than 2 allele choices.

                                                                                   Getz 2010   1
        o Alu elements are thought to be retro-transposons. Transposons are
             segments of DNA that jump from one location to another. If their jump
             happens in cells that lead egg and sperm (not autosomal cells, the germ line
             cells) then the mutation stays a part of that genetic line forever
                  RETROtransposons use an RNA intermediate in the process of
                     relocating themselves
                  Alu is considered to be a SINE, Short Interspersed Elements
                  RNA polymerase III is the enzyme that makes the RNA intermediate. A
                     reverse transcriptase then makes a DNA copy that gets inserted into a
                     region of chromosomal DNA.
                  An aside- HIV is a retrovirus because it starts as RNA and then
                     becomes DNA. The “retro” term is used to describe any nucleic acid
                     component that has the ability to go from RNA to DNA. This is
                     backwards to what we normally think about the way nucleic acids are
                     supposed to behave. The Central Dogma is that DNA leads to RNA, not
                     RNA leading to DNA.
        o For the DNA sequence that has an Alu element, there are two designations
             for the alleles
                  The “+”, plus, allele has the insert
                  The “-“, minus, allele does not
                  Therefore this type of inheritance pattern is referred to as “+/-“ when
                     looking at allele and genotype frequencies
        o Everybody who has the + allele in a specific region is genetically related. It is
             not statistically possible for alu elements to randomly jump into the exact
             same region twice.
        o Not having an alu element in a specific location does NOT mean two
             individuals are not related. It just means they don’t have an ancestor in
             common for that specific mutation. They may have a different ancestor in
             common. We can’t establish they have a common ancestor, though, based on
             this specific location in the human genome.
   Allele frequencies look at how often a specific allele shows up in populations.
   In many discussions, the “+” allele is designated as “p” and the “-“ allele is designated
    as “q” because Hardy-Weinberg math is often done using “p” and “q” to represent
        o Note: there are many regions in the human genome where there are more
             than two choices of alleles. This is due, in part, to SNP differences or other
             patterns that have been figured out in the last 10 – 20 years. The loci looked
             at by CODIS all have multiple possible alleles. “Locus” is a term used to refer
             to a specific location (“loc”) on a chromosome. Most often a locus location
             refers to where a gene is located, but it can also refer to a region whose
             genetic component is known about.

                                                                                 Getz 2010   2
   Given a total of 100%, if you add how often the p allele and the q allele show up,
    they will equal 1. 100% = 100 = 1
   So p + q = 1
   Applying the same genetic principles that let are explained by Punnett Squares, you
    can see how the equation used for Hardy-Weinberg equilibrium leads to the
    equation p2 + 2pq + q2 = 1
   Allele frequencies are referred to in terms of p and q (and if there are more possible
    alleles, they can also be referred to as r, s, t, etc.)
   Genotype frequencies are referred to in terms of p2, 2pq, or q2
                 parent one                   p                   q
                 parent two
                          p                  pp                  pq
                          q                  pq                  qq
   To get the HWE equation look at the results of a Punnett square:
         o pp, pq, pq, and qq
         o This becomes pp + pq +pq + qq
         o pp becomes p2
         o pq + pq becomes 2pq
         o qq becomes q2
   Adding up all of the possible genotype combinations possible, when the population
    is in Hardy-Weinberg equilibrium, the sum of the genotypes frequencies in the
    population will equal 1. Thus the most general equation seen for Hardy-Weinberg
    Equilibrium (HWE) is
            p2 + 2pq + q2 = 1
   For alu, the “p” allele is often written as + and the “q” allele is often written as –
   Therefore for Alu
         o The p2 genotype is actually written as +/+
         o The pq genotype is written as +/-
         o The q2 genotype is written as -/-
   For calculating genotype frequencies, you need to still double the pq value which is
    one reason H-W math is really confusing. To calculate genotype frequencies based
    on allele frequencies, you look at the population being tested, count up how often
    the p and q alleles show up, and then
         o Square the allele frequency for the p allele
         o Multiply the p allele frequency times the q allele frequency and then multiply
            by 2
         o Square the allele frequency for the q allele
         o The sum of these numbers should equal 1
   You can also go backwards. If you know a genotype frequency you can calculate the
    allele frequencies because it is an algebraic relationship.

                                                                               Getz 2010   3
      Keep in mind, HWE is in a theoretical world. It may NOT be reality. A statistical
       analysis called Chi-squared is used to figure out if a population is in Hardy-
       Weinberg equilibrium.
      Chi squared analysis looks at the probability of the calculated genotype frequencies
       based on the allele frequencies actually happening in nature. In most cases, the “null
       hypothesis” is rejected and the population is therefore not in HWE. It is actually a
       good thing for a population to not be in HWE. Hardy-Weinberg equilibrium is an
       artificial, “if the world was perfect”, type of situation.
           o At this point do not worry about the terms Chi-squared or null hypothesis.
               You’ll learn about them when you take statistics in college.

Alu, Pv92, lab
    Terms used to describe the controls do NOT talk about expected results
    Positive control:
           o The positive control should NOT necessarily yield a +/+ or +/- genotype
           o The positive control means the PCR reaction worked and a valid product was
               created therefore there is a band in the lane
    Negative control:
           o The negative control means NOTHING showed up in the lane because the
               “DNA” used is water
           o It is making sure there was no contamination in any of the reagents
               (chemicals) used
    The positive control worked if there are any bands in the lane. The result in that
       lane could be +/+, +/-, or -/-. These + and – designations don’t mean positive or
       negative, they mean the presence or the absence of the alu element.
    The negative control worked if there are NO bands in the lane. If there is a band
       then that means somebody made the control wrong or our reagents were
       contaminated with DNA.

World map of “p” allele frequencies
   Researchers figured out the p allele frequency for several populations
           o Why do you think they did not care about the q allele frequency?
   Using the p allele frequencies and the map, write down the allele frequency for each
      listed population. Please don’t let typos throw you off or waste your time.
   Look at the trends you see for where the p allele tends to show up
   Think about your genetic background- where mom came from, where dad came
      from, and hypothesize what your genotype will be. Do you think you will be +/+, +/-
      , or -/-
   If you were adopted, examine your phenotype and guess what you think your
      genotype will be for Pv92
   If you’ve done this lab before and therefore know your genotype, pick someone who
      does not look like you and hypothesize their genotype

                                                                                  Getz 2010   4

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