lab 7: pcr and pedigree analysis

B I O L O G Y 2 0 0 P C R & P E D I G R E E . 1 S U M M E R 2 0 2 0

Lab 7: PCR and Pedigree Analysis LAB GOALS:

• To understand how PCR amplification works and how it can be used to study DNA at the molecular level.

• To genotype your DNA at the bi-allelic pv92 locus for the presence or absence of Alu • To learn how to use pedigrees to solve a genetics problem.

Introduction to PCR Polymerase Chain Reaction (PCR) is the controlled enzymatic amplification of a DNA sequence of interest. PCR produces exponentially large amounts of a specific piece of DNA using only trace amounts of original (template) DNA. The invention of PCR techniques by Kary Mullis in 1983 opened the door to a new world of genetics research. Many techniques used before PCR were labor intensive, time consuming, and required a high level of technical expertise. Additionally, working with trace amounts of DNA made it difficult for researchers in biological fields such as pathology, zoology, ecology, pharmacology, etc to incorporate genetics into their research schemes. PCR technology changed all this and allowed for major increases in our understanding of genetics. One of the main reasons PCR is such a powerful tool is its simplicity and specificity. All that is required are reaction buffers, the four deoxyribonucleotide triphosphates (adenine, guanine, thymine, and cytosine), DNA polymerase, two DNA primers, minute quantities of the template DNA strand, and a thermocycler. PCR amplification requires the presence of at least one DNA template strand. This template strand comes from the organism of interest. In our case, the DNA template strand will be isolated from your own cheek cells!

How does PCR work? PCR involves a repetitive series of cycles, each of which consists of DNA template denaturation, primer annealing (binding), and extension of the annealed primer by DNA polymerase. During DNA template denaturation, the original double-stranded DNA template is separated into two single-strands, thus allowing access to the unpaired bases by the primers. What are the primers? The primers used in PCR are designed and synthesized in a laboratory to have specific sequences of nucleotides such that they can anneal upstream and downstream of the DNA sequence of interest. During primer annealing, these primers bind to specific regions on each single-stranded DNA template. Once the primers are annealed, DNA polymerase adds deoxynucleotides to the 3’ end of the primer. The DNA polymerase used in PCR must be thermally stable because the reaction cycles between temperatures of 60°C and 94°C. Thus, a DNA polymerase from a thermophilic bacterium (Thermus aquaticus) that lives in high-temperature steam vents is used in PCR. This DNA polymerase is called Taq. Following DNA template preparation, the template DNA, primers, DNA polymerases (Taq), the four deoxynucleotides, and reaction buffer are mixed in a single micro test tube. The micro test tube is then placed into a thermal cycler, a machine that can be programmed to rapidly heat and cool across extreme temperature differences. The first step of PCR (the denaturation step) is to heat the sample to 94°C. At this high temperature, the hydrogen bonds that hold DNA template strands together are broken causing them to separate (denature). Next, the thermal cycler rapidly cools to 60°C, allowing the primers to bind to the separated DNA strands (the annealing step). Why do the primers bind to the DNA strands rather than the DNA strands binding to each other once again? The reason is that the primers are added in excess so that there are more primers than DNA templates, making it more likely that a single-stranded DNA template will bind to a primer than to another DNA template. During the


last step of PCR, the sample is heated to 72°C and Taq DNA polymerase adds deoxynucleotides to the primers, making complete copies of each template DNA strand (the extension step). These three steps—denaturation, annealing, and extension—comprise a complete thermal cycle. Usually, thermal cycling continues for 30-40 cycles. After each thermal cycle, the number of DNA template strands doubles, resulting in an exponential increase in the number of DNA template strands. After 40 thermal cycles, there will be 1.1 x 1012 more copies of the original number of template DNA molecules!!

Recall that one of the amazing things about PCR is its specificity. PCR generates DNA of a precise length and sequence. On the first cycle, the two primers anneal to the original template DNA strands at opposite ends at the sequence of interest and on opposite strands. After the first complete thermal cycle, two new strands are generated that are shorter than the original template strands but still longer than the length of the DNA sequence of interest. It isn’t until the third thermal cycle that fragments of the precise length are generated (see figure below).


Agarose Gel Electrophoresis To determine if you have the Alu insert in your genome, you will separate the DNA fragments amplified during PCR using gel electrophoresis.

Electrophoresis separates DNA fragments according to their relative sizes (number of base pairs (bp)). This is done by loading the DNA fragments into an agarose gel slab, which is placed into a chamber filled with a conductive buffer solution. A direct current is passed between wire electrodes at each end of the chamber. Since DNA fragments are negatively charged, they will be drawn toward the positive pole and repelled by the negative pole when placed in an electric field. The matrix of agarose gel acts as a molecular sieve through which smaller DNA fragments can move more easily than larger ones. Over a period of time, smaller fragments will travel farther than larger ones. Fragments of the same size stay together and migrate in what appears as a single “band” of DNA in the gel. Figure 1 below shows an example of an agarose gel with stained DNA fragments separated by size. The bands in lane 1 are the molecular ladder which is a sample containing DNA fragments of known sizes. In this case, the molecular mass ruler contains 1,000 bp, 700 bp, 500 bp, 200 bp, and 100 bp fragments. The molecular ladder is used to determine the size of unknown DNA fragments and to track the position of expected fragments. In addition to the molecular ladder, you will also run known controls in your gel. The controls are: homozygous positive for the Alu insert (+/+), homozygous negative for the Alu insert (–/–), and heterozygous for the Alu insert (+/–). Figure 2 indicates the expected size of the DNA fragments for each control. Since DNA is colorless, the position of the DNA fragments is not visible in the gel until the gel is stained. Unstained visual examination of the gel after electrophoresis indicates only the position of the loading dyes, NOT the positions of the DNA fragments. DNA fragments are visualized by staining with a DNA-binding stain. The dye molecules are positively charged and have a high affinity for DNA. Therefore, these dye molecules strongly bind to the DNA fragments and allow the DNA bands within the gel to become visible when exposed to ultra violet light.

Figure 1: Example of a gel with the molecular ladder banding pattern in column 1. Note the varying widths and darknesses of the various bands.

*This is a great example of a publication-quality gel. Your gel is likely to be less clear than this, and it may take some effort to read it the first time.


Figure 2: PV92 Alu insertion controls. Three genotypes are common at the PV92 locus. Each genotype will have a different banding pattern. A control is provided that should give results that mimic each genotype.

PV92 Genotype Size of fragment

+/+

941 bp

–/–

641 bp

+/–

941 bp & 641 bp

Laboratory Instructions Agarose Gel Electrophoresis Overview This week in lab, you will analyze your amplified DNA from last week using agarose gel electrophoresis. Electrophoresis separates DNA fragments according to their relative sizes (number of base pairs (bp)).

WEAR GLOVES WHILE WORKING WITH YOUR GELS

1. Find your tubes, including loading dye (LD), molecular ladder (MR), homozygous (+/+) control, homozygous (–/–) control, and heterozygous (+/–) control. Each lab bench is also provided with one agarose gel and one electrophoresis chamber. You and your lab partners will share the above items, using one gel to run all of your DNA samples.

2. Obtain your amplified DNA from last week’s lab.

3. Using a P20 micropipet, add 10 µl of loading dye to each PCR tube, including the controls. You DO NOT need to add loading dye to the molecular marker because it already contains dye. Mix gently by pipetting up and down a few times. Place in the ice bucket. Note: This loading dye is blue or orange, but is different from the large bottle of blue staining solution that you will use later. Don’t mix these up.

4. Using a clean tip for each sample, load the samples into the wells of the gel following the order and loading volume shown in the chart below. Try not to get bubbles in the wells. If there are bubbles, you can “chase” them out with the tip of your pipet. After you’ve finished loading your gel, you will have two empty lanes

Alu

Alu

Alu


5. Fill in the student names in the chart. This step is VERY IMPORTANT! If you do not do it, you won’t be able to find your DNA!

LANE SAMPLE Amount of loading dye to add to sample

Amount to load into the gel well

1 MMR NONE 10 µl

2 Homozygous (+/+) control 10 µl 10 µl

3 Homozygous (–/–) control 10 µl 10 µl

4 Heterozygous (+/–) control 10 µl 10 µl

5 Student 1; name: 10 µl 40 µl (all)




6. Secure the lid on the gel box, ensuring that the red wires on the lid match up with the red tape on the gel box. Connect the electrical leads to the power supply.

7. Turn on the power supply and ensure it is set for ~100 V. Run the gel for 30-45 minutes. You want to run the gel far enough that you can see a difference between band sizes, but not so far that the DNA leaves the gel. If the blue part of the loading dye is about half-way down the gel then you have probably run the gel long enough to see results.

8. After running, turn off the power supply and remove the lid from the gel box. Very carefully remove the tray from the gel box. The gel is slippery and can easily slide off! Nudge the gel off the gel tray with your thumb and gently slide it into the plastic staining tray.

9. Staining: before class, course staff mixed DNA-binding stain into the agar called SybrSafe. While the agar is running, the dye will bind to DNA molecules. When viewed with visible light, DNA and the dye are not visible. When viewed under ultraviolet (UV) light the dye-DNA complex fluoresces green.

10. Wearing gloves, remove the gel cradle from the electrophoresis box. Transfer to the UV transilluminator box at the rear of the lab room.

a. Caution: i. You must wear gloves while handling the stained agar.

ii. UV light is a form of ionizing radiation. The viewing screen has a UV filter. Only view through this screen.

11. Once you can see visible bands, you are ready to make a record of your gel. Place your gel on a light background so the bands are easily visible. Also make sure that the correct side is facing up (refer to your chart)! Draw your gel, using the gel schematic on the following page.


Interpretation Compare your sample lane with the control lanes using the molecular ladder as a size reference. How do your DNA bands compare to the controls? By comparing your DNA banding pattern to these controls, you should be able to determine whether you have the Alu sequence at the PV92 locus on both, one, or neither of your chromosome 16 homologues. Remember that this Alu sequence is inserted into a noncoding region of the PV92 locus on chromosome 16. It does not code for any protein sequence and it is not related to a particular disease.

Complete the discussion questions and hand them in to your TA.

Clean-Up Instructions • Pipet tips go into the lab glass. All of the tubes can go in the trash. • Staining & destaining trays, plastic beakers, funnel, electrophoresis chamber: Rinse

with hot water • Ensure power supply is turned ‘off’ • Sketch gel, and then discard gel in trash • Disinfect benchtop • Wash your hands


Gel Drawing 1) Fill in the expect results for Lanes 1-4 before you start the electrophoresis. 2) Fill in the results from other lanes after you determine any genotypes you can.

3) If you do not successfully find your genotype, fill in what you would have seen (pick your own genotype).

1 2 3 4 5 6 7 8 9 10

MM

R

(+/+) control

(-/-) control

(+/-) control


PCR / Gel Electrophoresis Questions Names ___________________________________________________________ Section __________

1. Recall that after you extracted your DNA a few weeks ago, we ran a PCR to analyze your DNA. a. What ingredients did we add to each PCR tube in order for the PCR to work?

What was the purpose of each ingredient?

b. Even though the TA rarely makes mistakes, pretend that they programmed the PCR machine

incorrectly. Rather than programming the denaturation cycle for 94°C, they set it for 54°C. What is one likely result of this mistake?

2. Imagine that you accidentally hooked up the electrodes for your gel electrophoresis box incorrectly

so that the positive and negative poles have been swapped. You electrophorese the gel for 30 minutes but when you stain the gel, you can’t find any DNA bands in any of the lanes. Where is the DNA and why don’t you see bands?

3. On your gel drawing, write the appropriate student name next to each lane. Label each banding

pattern as homozygous positive (+/+), heterozygous (+/-), or homozygous negative (-/-). Please staple the drawing of your gel to this page.

4. After discussing the particular demographics of the pv92 Alu insertion with your TA: a. Does your genotype make sense? b. If a classmate were of purely East Asian descent, would a -/- genotype be possible at this locus? Why or why not?

B I O L O G Y 2 0 0 P C R & P E D I G R E E . 1 0 S U M M E R 2 0 2 0


Introduction to Pedigree Analysis: Typically when we talk about phenotypes, you think of eye color or hair color, but using molecular techniques, such as PCR, some differences in DNA can be resolved and visualized by gel electrophoresis. The banding pattern you see on the gel is an individual's molecular phenotype, which also tells you that individual's genotype. Today, you will use "typical" phenotypes and molecular phenotypes to solve a pedigree problem. In Biology 180 and other courses, you have thought about the inheritance of genes in Drosophila and other organisms. Why can’t we study humans in the same way as Drosophila? With Drosophila, we can mate two types of flies to see the different phenotypes of progeny they create and in what ratios. However, it would be unethical to force the mating of certain humans simply to learn about their genes. Therefore, human genetic analysis requires the information stored in a family tree – or in genetic terms, a pedigree. As you work this pedigree problem, you will be considering both “typical” and molecular genotypes. Use them both to solve the problem!

HUMAN PEDIGREE ANALYSIS Were Nat and Boris switched at birth? In the following pedigree, there are three generations of a family. Fred and Ethyl Menendez have three daughters: Adelle and Briana are identical twins and they have a younger sister, Carly. Each daughter is married and has one son. Nat and Boris were born on the same day in the same hospital. And that is where the potential problem arose.

In the news during the summer of 1993, a woman who had been a nurse in a hospital in Florida nearly 15 years previously, admitted on her deathbed that she deliberately switched two newborn babies at birth. A different nurse, who worked in the nursery where Boris and Nat were born, heard this story and began to worry that she may have inadvertently mixed up those two babies. Adelle and Briana both delivered their sons on the same day and because the two moms looked exactly alike, the nurse felt it was possible that she had gotten confused when she took the two babies to their mothers for their

Fred Ethyl

Wei-Shiung Adelle Briana

Boris

Luigi Carly

Olaf Nat

Gustav

= identical twins


first feedings. She contacted the families and now the families have come to a genetic counselor to try and determine if there had indeed been a mix-up.

To confirm whether or not a mix-up occurred, you will examine the results from a series of five tests conducted on the members of this family. More than one test may provide proof one way or the other. Obtain results from each test in order and fully examine one trait before you move onto another test. For some traits you may not be able to fully assign genotypes for each individual until you have determined the true parentage of Boris and Nat. You should check ALL traits to make a confident case of parentage.

Work with your Lab Partners to Complete the Following Steps 1. For each test, review how the trait is inherited (dominant or recessive, autosomal or sex-linked),

then go to your TA and get the test results for just that trait. Record the phenotypes on the pedigrees on the next page. (Your TA will go through the first trait (tongue-rolling) to demonstrate).

2. Try out possible genotypes. Be sure to use the standard allele symbols (from chart on board). Remember to indicate unknown alleles with a “?” Record the completed results on the pedigrees for reference.

3. When you have found the best solution for the particular trait, record the genotypes under the symbols of each individual in the pedigree. If you are uncertain, have the TA check your results.

4. As you consider each trait, decide whether or not there is evidence that the mix up occurred. Indicate if the trait is informative in the column at the bottom of the chart. You need to consider all the traits and decide for each whether or not they are informative. If one trait is informative that should not stop you from ascertaining the status of other traits.

5. Move on to the next trait, review its inheritance, and then get the test results from the TA. Wipe the acetate copy of the pedigree using the ethanol provided and generate a solution for this second trait.

6. Continue through all of the tests. Your TA will circulate through the lab to help. 7. You will receive test results for each member of the family for the following traits:

• Rh blood type

• ABO blood type

• Colorblindness

• Hemophilia

• MAM02 – you have a picture of a gel with PCR products amplified from the “MAM02” locus for each member of the pedigree. It is your job to determine the genotype of each individual.

• X-linked: For this pedigree, combine the pedigree information for the two X-linked traits. 8. When you have finished this exercise, you will have decided whether the baby boys were switched

at birth or not. In addition, you may uncover other abnormalities in this family and hopefully solve them.

9. Complete and turn in the pedigree analysis questions on pages 9.25-9.26.


Fred Ethyl

Wei-Shiung Adelle Briana Luigi Carly

Olaf Nat

Gustav

Hemophilia

Fred Ethyl


Olaf Nat

Gustav

Color Blindness

Boris

Boris


Fred Ethyl


Olaf Nat

Gustav

Rh Factor

Fred Ethyl


Olaf Nat

Gustav

ABO Blood Type

Boris

Boris


Fred Ethyl


Olaf Nat

Gustav

MAM02 Polymorphic Locus

Fred Ethyl


Olaf Nat

Gustav

X-linked: combine the pedigrees for the two X-linked traits

Boris

Boris


Pedigree Analysis Questions

Names ___________________________________________________________ Section __________

1. Were Nat and Boris exchanged at birth?

2. Which test(s) were informative? 3. How did Boris get his special karyotype? Which parent could be responsible for the extra

chromosome? Draw the starting cell and the products of meiosis I and II in that parent.

4. Which allele for color blindness is linked to which allele for hemophilia on Carly’s two X

chromosomes? On the chromosomes below, indicate the linkage arrangement. (Hint— use the provided X-linked pedigree)

Another way to indicate X chromosome linkage is to write the alleles as superscripts on an X. Show Carly’s X chromosome genotypes below.

X X


5. What is the probability that Wei-Shung (IAIBrr) and Adelle (IAiRr) could produce a child with Wei-Shung's blood phenotype (AB, Rh—)? Show your work.

6. Does MAMO2 appear to segregate as though tightly linked to any of the other loci we have

considered today? To determine this you will need to compare MAMO2 with each trait.

7. On which chromosome is the MAMO2 locus?

8. Does this linkage information clarify Boris’ genotype at any locus? If so, show the clarification.

lab 7: pcr and pedigree analysis

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