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% of the human genome does not encode proteins. Many DNA 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. You will pick a number from a box to use in this lab. This number will be used as your ID throughout the 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

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.

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.

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

Materials needed (per student):

Saline solution (0.9% NaCl), 10 ml

10% Chelex®, 100 µl

Paper cup

Microfuge tubes, large (1.5 ml) and small (0.6 ml)

Micropipettes and tips

Cup of ice

Microcentrifuge

Boiling water bath or Thermal cycler

Methods:

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

2. Expel saline solution into the paper cup.

3. Swirl to mix your buccal cells with saline and transfer 1 ml (1000 µl) of the liquid to a large microfuge (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. (Supernatants and remaining student samples are allowed to be disposed down lab drains)

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 small microfuge (0.6 ml) tube. Add 100 µl of 10% Chelex. Shake well to mix.

8. Boil cell sample for 10 minutes. Use boiling water bath or thermal cycler programmed for 10 minutes at 99°C. (the file in our thermal cycler used for this step is called denaturation). Samples are boiled to lyse the cell and nuclear membranes 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, vigorously shake the tube for 5 seconds. Place in a balanced configuration in a microcentrifuge, and spin for 1 minute.

10. Transfer 30 µl of supernatant (containing the DNA) to clean small microfuge (0.6 ml) tube. Avoid cell debris and Chelex beads. This sample will be used for setting up the two PCR reactions.

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

12. Paper cup should be discarded in a biohazard bag.

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 micropipette 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 loading dye solution.

· Each PCR bead contains reagents when brought to a final volume of 25 µl the reaction; 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:

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. Obtain two tubes with Ready-to-Go Beads™ from the instructor. Label caps of the two tubes with your ID number for this lab, so your results will be anonymous. In addition to this number, label one tube A (for Alu) and the other V (for VNTR) on the caps as well. (eg: 5A and 5V)

2. Use a micropipette 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 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 (Alu PV92) 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

Part III: OBSERVATION OF aLU AND VNTR POLYMORPHISMS THROUGH agarose Gel Electrophoresis

Materials needed (for entire class):

PCR amplified tubes of Alu and VNTR polymorphism

100 bp DNA ladder

Agarose

1X TBE buffer

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 seven students. Use the other gel for VNTR PCR tubes. Your instructor will ask you to pour additional gels depending on the number of students in class at the time.

Alu gel:

Load 8 µl of 100 bp 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 8 µl of 100 bp ladder into lane 1 of gel.

Use lanes 2-7 to load 15µl PCR sample/loading dye mixture for samples labeled 1-6.

Run the gel and stain with ethidium bromide. Take a picture of the gel and obtain a hard copy.

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 first lane of the gel containing 100 bp ladder fragments. Working from the well, locate the bands corresponding to fragments of sizes 10,000?bp, 1,000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300 bp, 200bp and 100 bp. Additional 50 bp band may be faintly visible.

c. Scan one lane at a time. If only one band is visible, compare its migration to that of the 100 bp ladder. If the PCR product migrates slightly ahead of the 700 bp ladder fragment, then the person is homozygous for the PV92 Alu insertion (+/+). If the PCR product migrates slightly ahead of the 400 bp ladder band, 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 700 and 400 bp ladder fragments. 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. The person is heterozygous for the PV92 Alu insertion (+/-).

e. It is common to see an additional band lower on the gel. This diffused (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 the lowest part of your gel.

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 or incompletely amplified PCR 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 with a non-complementary loop sticking out.

2. Write the Genotype distribution and calculate genotype frequency for Alu PV92 for class:

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.