Human Dna Polymorphism

HUMAN DNA POLYMORPHISM

Although DNA from various individuals is more alike than different, many regions of human chromosomes exhibit a great deal of diversity. Such variable sequences are termed polymorphic, meaning many forms, and are used for forensic identification, diagnosis of genetic disease and paternity testing. An estimated 98.5% of the human genome does not encode proteins. Many polymorphisms are located within these non- coding regions.

Several kinds of polymorphisms have been observed in the human genome. In this lab you will develop two kinds, Alu insertion polymorphism and VNTR (Variable number of tandem repeats) polymorphism. Both of these polymorphisms will be generated using the polymerase chain reaction.

You will work individually for generating both kinds of polymorphisms using your own DNA in this lab. Specific markers used in this lab appear to have no known relationship to disease status, sex determination or any other phenotype. Still, to protect against the unforeseen possibilities of misuse of student data, the confidentiality of student genotypes will be maintained by identifying student samples only by numbers. Your instructor will assign you a number to use in this lab.

DNA isolated from your cheek cells will be used to develop both kinds of polymorphisms by PCR. You will set up two PCR reactions from the same DNA, one for Alu polymorphism and the other for VNTR polymorphism.

Human PV-92 Alu insertion Polymorphism

Copyright: Dolan DNA Learning Center, Cold Spring Harbor Laboratory http://www.geneticorigins.org

Alu elements are located in the non-coding regions of the primate genomes. Apes, monkeys and humans are considered as primates, since they all share a common evolutionary branch that separated from other vertebrate groups more than 65 million years ago. All Alus are approximately 300 bp in length and derive their name from a single recognition site for the restriction enzyme AluI, AGCT, located near the center of the Alu element. Alu elements are abundant in the human genome. Human chromosomes contain about 1,000,000 Alu copies, accounting for 10% of the total genome.

Alu is an example of a so-called "jumping gene" – a transposable DNA sequence that "reproduces" by copying itself and inserting into new chromosome locations. Alu is classified as a retroposon, because it is thought to require the retrovirus enzyme reverse transcriptase (rt) enzyme to make a mobile copy of itself.

1 Once an Alu inserts at a chromosome locus, it can copy itself for transposition, but there is no evidence that it is ever excised or lost from a chromosome locus. So, each Alu insertion is stable through evolutionary time. Each is the "fossil" of a unique transposition event that occurred only once in primate evolution. Like genes, Alu insertions are inherited in a Mendelian fashion from parents to children. Thus, all primates showing an Alu insertion at a particular locus have inherited it from a common ancestor. This is called identity by descent.

An estimated 500-2,000 Alu elements are restricted to the human genome. The vast majority of Alu insertions occurs in non-coding regions, and is thought to be evolutionarily neutral. Most Alu mutations are "fixed," meaning that both of the paired chromosomes have an insertion at the same locus (position). However, a number of human-specific Alus are dimorphic – an insertion may be present or absent on each of the paired chromosomes of different people. These dimorphic Alus inserted within the last million years, during the evolution and dispersion of modern humans. These dimorphisms show differences in allele and genotype frequencies between modern populations and are tools for reconstructing human prehistory.

This experiment examines PV92, a human-specific Alu insertion on chromosome 16. The PV92 genetic system has only two alleles indicating the presence (+) or absence (-) of the Alu transposable element on each of the paired chromosomes. This results in three PV92 genotypes (++, +-, or --). The + and - alleles can be separated by size using gel electrophoresis.

VNTR POLYMORPHISM

A special type of human polymorphism, termed VNTR (variable number of tandem repeats) is a composed of stretches of DNA that contain a core sequence repeated over and over in tandem (one after another) on the chromosome. VNTRs are highly polymorphic minisatellite markers because the number of repeats varies from one individual to another.

2 In this lab we will generate polymorphism in the VNTR designated pMCT118 located in the non-coding region of chromosome 1. It has a repeat unit of 17 base pairs. Most individuals have between 14 and 40 copies of the repeat unit on both the maternal and paternal copies of the chromosome 1. The maternal and paternal copies of chromosome 1 usually have different number of repeats, as do the chromosomes from different individuals. Because the VNTR locus has more than 29 different alleles, a panel of test subjects will generate a variety of different genotypes.

PCR amplification of VNTR polymorphisms is complicated by the presence of repeated units. Therefore amplification of these types of markers can greatly benefit from a “hot start” method, where one of the reagents is added during the first denaturing cycle. We will perform a variation of a hot start method in this lab by adding the PCR tubes to the thermal cycler when the block has reached 95 o C.

PART I: CHEEK CELL DNA ISOLATION BY SALINE MOUTHWASH

(Method adopted from Gen 303 Lab manual, Clemson University)

Materials needed (per student): Saline solution (0.9% NaCl), 10 ml 10% Chelex®, 100 µl 15 ml test tube, polypropylene Paper cup 1.5 ml test tubes, polypropylene 1 ml transfer pipet or 100-1,000 µl micropipet and tip Cup of ice Microcentrifuge Boiling water bath, heat block, or Thermal cycler

Methods:

1. Pour 10 ml of the saline solution (0.9% NaCl) into mouth and vigorously swish for 30 seconds.

2. Expel saline solution into a paper cup.

3. Swirl to mix cells in the cup and transfer 1 ml (1000 µl) of the liquid to 1.5 ml tube.

4. Place your sample tube, together with other student samples, in a balanced configuration in a microcentrifuge, and spin for 1 minute.

5. Carefully pour off supernatant into paper cup or sink. Be careful not to disturb the cell pellet at the bottom of the test tube. A small amount of saline will remain in the tube.

3 6. Resuspend cells in remaining saline by pipetting in and out. (If needed, 30 µl of saline solution may be added to facilitate resupension.)

7. Withdraw 30 µl of cell suspension and add to a 0.5 ml tube. Add 100 µl of 10% Chelex. Shake well to mix.

8. Boil cell sample for 10 minutes. Use boiling water bath, heat block, or program thermal cycler for 10 minutes at 99°C. Then, cool tube briefly on ice. Samples are boiled to lyse the cells and liberate the chromosomal DNA. The Chelex binds to metal ions that are released from the cells that inhibit the PCR reaction.

9. After boiling, shake tube. Place in a balanced configuration in a microcentrifuge, and spin for 1 minute.

10. Do not disturb the tube. The supernatant containing the DNA will be used for setting up the two PCR reactions in Part II. Chelex and cell debris will be at the bottom of the tube. Do not use this pellet.

11. Optional: Store your sample on ice or in the freezer until ready to begin Part II.

PART II: AMPLIFICATION OF PV92 ALU ELEMENT AND PMCT118 VNTR BY PCR

Materials needed (per student):

Your cheek cell DNA extracted in part I Two tubes of Ready-to-Go Beads™ 1-20 µl micropipet and tips Thermal Cycler Ice in a container For Alu polymorphism: PV92B primers/loading dye mix (22.5 µl)

For VNTR polymorphism:pMCT118 primers/loading dye mix (22.5 µl)

 Primers/loading dye mix for both kinds of polymorphisms includes both primers (0.25 pmol/µl of each primer) in cresol red solution.

 Each PCR bead contains reagents when brought to a final volume of 25 µl the reaction contains 1.5 units of Taq polymerase, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2 and 200 µM of each dNTP.

Methods:

4 Set up two PCR reactions, one for Alu and one for VNTR with your cheek cell DNA. Be sure to use the respective primers for each kind of polymorphism.

Set up reaction tubes on ice and keep all tubes on ice until ready to put in the thermal cycler. Work quickly and initiate thermal cycling as soon as possible after mixing PCR reagents.

1. Label caps of two 0.5 ml microfuge tubes with your ID number for this lab, so your results will be anonymous. Label one tube A (for Alu) and the other V (for VNTR) on the caps as well.

2. Use a micropipet with a fresh tip to add 22.5 µl of the respective primer/loading dye mix to each of the PCR tubes containing a Ready-To-Go PCR Bead. Tap tube with finger to dissolve bead.

3. Use fresh tip to add 2.5 µl of supernatant containing your DNA from Part I to each reaction tube, and tap to mix. Pool reagents by pulsing in a microcentrifuge or by sharply tapping the tube bottom on lab bench.

4. Start the pre-programmed file in the thermal cycler. To perform the variant hot-start method, place tubes in the thermal cycler, after the block has reached the denaturation temperature,

The thermal cycler should run the following: One time initial denaturation: 3 min at 95o C 30 cycles of  Denaturation: 30 sec at 94o C  Primer annealing: 30 seconds at 65o C  Primer extension: 30 sec at 72o C. One time final extension: 7 min at 72o C The block will cool down to 4o C

DNA ANALYSIS BY AGAROSE GEL ELECTROPHORESIS

To carry out agarose gel electrophoresis, the class will work together. Cast two 2% agarose gels. One gel is sufficient to run Alu PCR samples of the entire class. Use the other gel for VNTR PCR tubes.

Alu gel: Load 6 µl of the Bench top 100 bp DNA ladder into lane 1 of gel. Use lanes 2-7 to load 15µl PCR sample/loading dye mixture for samples labeled 1-6.

VNTR gel: Load 6 µl of the Bench top 100 bp DNA ladder into lane 1 of gel. Use lanes 2-7 to load 15µl PCR sample/loading dye mixture for samples labeled 1-6.

5 Interpreting the class PV92 Alu insertion polymorphism gel

1. Observe the photograph of the stained gel containing your sample and those from other students. Orient the photograph with the sample wells at the top. Interpret the band(s) in each lane of the gel:

a. Scan across the photograph to get an impression of what you see in each lane. You should notice that virtually all student lanes contain one to three prominent bands.

b. Now locate the lane containing the pBR322/BstN I markers. Working from the well, locate the bands corresponding to each restriction fragment: 1,857bp, 1,058 bp, 929 bp, 383 bp, and 121 bp (may be faint).

c. One band visible. Compare its migration to that of the 929-bp and 383-bp bands in the pBR322/BstN I lane. If the PCR product migrates slightly ahead of the 929 bp band (lane 4), then the person is homozygous for the PV92 Alu insertion (+/+). If the PCR product migrates well behind the 383 bp band (lane 2), then the person is homozygous for the absence of the PV92 Alu insertion (-/-).

d. Two bands visible. Compare migration of each PCR product to that of the 929-bp and 383-bp bands in the pBR322/BstNI lane. Confirm that one PCR product corresponds to a size of about 715 bp and that the other PCR product corresponds to a size of about 415 bp (lanes 1 and 3). The person is heterozygous for the PV92 Alu insertion (+/-).

e. It is common to see an additional band lower on the gel. This diffuse (fuzzy) band is "primer dimer," an artifact of the PCR reaction that results from the primers overlapping one another and amplifying themselves. Primer dimer is approximately 50 bp, and should be in a position ahead of the 121 bp marker.

a. Additional faint bands at other positions occur when the primers bind to chromosomal loci other than PV92 and give rise to "nonspecific" amplification products.

b. A faint band at approximately 1130 bp observed only in heterozygous individuals is also an artifact. It is caused by heteroduplex formation between complementary regions of the two alleles.

2. Genotype distribution is simply the distribution of the three possible genotypes +/ +, +/- and -/-. Example: a class of 100 students list their genotype distribution as follows: +/+ 20 +/- 50 -/- 30. Write the genotype distribution in your class.

1. An allele frequency is a ratio comparing the number of copies of a particular allele to the total number of alleles present. Consider the above example of class a of 100 students. Since humans are diploid, the total number of alleles in the class is 2 x 100

6 = 200. The allele frequency for PV92+ is: 2 x 20 (homozygotes) + 50 (heterozygotes) / 200 = 90 / 200 = 0.45 Likewise, the allele frequency for PV92- is: 2 x 30 (homozygous) + 50 (heterozygotes) / 200 = 110 / 200 = 0.55. Using the genotype distribution from your class, calculate the frequencies of the + and - alleles of PV92.

Interpreting the class VNTR polymorphism gel

1. Plot the distance vs. log size for the pBR322/BstN I marker bands in lane 1.

2. Use the above graph to determine the size of the alleles in each individual.

3. How many alleles are present in the entire class?

4. Of the two kinds of human polymorphism markers (Alu and VNTR) which one is more polymorphic and provides more information? Why?

Homework

1. Determine the genotype distribution and allele frequency for Alu PV 92 in the class.

2. What can you say about any person in the class who has at least one + allele?

3. What are primer dimers? Were there any primer dimers in your gels? What do primer dimers tell you about your PCR reaction?

4. Do you think these two polymorphisms alone would be useful in paternity testing or linking a suspect with a crime?

5. VNTR polymorphism was originally developed using Southern hybridization. What are the differences in developing VNTR polymorphism using Southern hybridization and PCR?