This experiment uses Polymerase Chain Reaction (PCR) to amplify a 440 base-pair segment of your own mitochondrial DNA (mtDNA) from cheek cells, then visualizes the result using agarose gel electrophoresis. PCR is essentially a DNA photocopier — it takes a tiny amount of DNA and makes billions of identical copies in just a few hours.
| Step | Temperature | Time | What Happens |
|---|---|---|---|
| Denaturation | 94°C | 30 sec | Heat breaks hydrogen bonds between base pairs → double-stranded DNA (dsDNA) separates into two single strands |
| Annealing | 58°C | 30 sec | Temperature drops → primers bind (anneal) to their complementary sequences on each single strand via hydrogen bonds |
| Extension | 72°C | 30 sec | Taq polymerase adds dNTPs from 5’→3’, extending the primer into a full complementary copy of the template |
Memory trick — Hot, Warm, Just Right: 94°C = scorching hot (unzips DNA), 58°C = cool (primers snap in), 72°C = warm (Taq works best, same temp as its natural hot spring)
Q1. At what temperature is Taq DNA polymerase most active? ✅ 72–75°C Taq polymerase comes from Thermus aquaticus, which lives in hot springs at ~72–75°C. This is the optimal temperature for extension. At 94°C it doesn’t function (just denatures DNA); at 58°C it’s too cool for efficient synthesis.
Q2. The two essential components added in addition to DreamTaq Master Mix are: ✅ Primers and DNA template The Master Mix already contains Taq polymerase, dNTPs, MgCl₂, and buffer. The two things YOU add are the specific primers (to define what region gets amplified) and your DNA template (your actual cheek swab DNA).
Q3. True or False: The two primers for one PCR reaction should be complementary to each other. ✅ FALSE If the primers were complementary to each other, they would bind to each other (forming “primer dimers”) instead of binding to the template DNA. The two primers must each be complementary to OPPOSITE strands at OPPOSITE ends of the target region — not to each other.
Q4. True or False: The direction of synthesis of the new DNA strand in each cycle is from 5’ to 3’ end. ✅ TRUE DNA polymerase (including Taq) can only add nucleotides to the 3’ end of a growing strand. Synthesis always proceeds in the 5’→3’ direction. This is a fundamental rule of DNA replication.
Q5. Which of the following are true regarding mitochondrial DNA? (Choose all that apply) ✅ A: The mitochondrial genome contains 37 genes. ✅ D: There are more copies of mt genes than nuclear genes per cell.
Purpose: PCR amplifies a specific, defined segment of DNA exponentially, allowing researchers to produce billions of copies from a tiny, even degraded starting sample. This makes it possible to detect, study, or sequence DNA that would otherwise be too scarce to work with.
Mechanism:
a) Essential components of a PCR reaction: - Template DNA (the DNA to be copied) - Two primers (short DNA sequences flanking the target region) - Taq DNA polymerase (heat-stable enzyme that synthesizes new DNA) - dNTPs (A, T, G, C building blocks for new strands) - MgCl₂ (cofactor required for Taq polymerase activity) - Buffer (maintains proper pH and ionic conditions)
b) What kind of DNA can be used as template? Does it have to be pure? Almost any double-stranded DNA can be used as template — genomic DNA, mitochondrial DNA, plasmid DNA, even ancient or degraded DNA. It does NOT need to be pure. Chelex extraction from cheek cells produces crude DNA with cellular debris, but PCR still works because the primers are highly specific to the target sequence.
c) Basic properties of PCR primers and how they are designed: - Primers are short single-stranded DNA sequences (~18–25 nucleotides) - Each primer is complementary to ONE strand of the DNA template - The two primers bind OPPOSITE strands at OPPOSITE ends of the target region - They are NOT complementary to each other - They define the boundaries (and therefore the size) of the PCR product - The 5’→3’ orientation of each primer points inward toward the other primer
d) Why must there be primers in the reaction? DNA polymerase CANNOT start synthesis from scratch — it can only ADD nucleotides to an existing strand. Primers provide the starting 3’-OH group that Taq polymerase needs to begin extending. Without primers, no new DNA can be synthesized.
e) What is special about the polymerizing enzyme used in the reaction? Taq polymerase is thermostable — it can withstand the 94°C denaturation temperature without being destroyed. Normal DNA polymerases would unfold and lose function at this temperature. Taq comes from Thermus aquaticus, a bacterium that naturally lives in ~72–75°C hot springs, so high temperatures are its normal operating environment. Limitation: no proofreading → higher mutation rate (~1/10,000 bases).
f) The three basic steps, temperatures, and purposes: 1. Denaturation: 94°C, 30 sec → Breaks hydrogen bonds between base pairs; separates dsDNA into two single strands (template strands) 2. Annealing: 58°C, 30 sec → Temperature drops; primers bind via hydrogen bonds to their complementary sequences on each single-stranded template 3. Extension: 72°C, 30 sec → Taq polymerase binds at the primer’s 3’ end and adds dNTPs in the 5’→3’ direction, synthesizing a new complementary strand
g) Is the amplification linear or exponential? Exponential. Each cycle doubles the number of DNA molecules. After n cycles: 2ⁿ copies. After 30 cycles: 2³⁰ ≈ 1 billion copies from a single template molecule.
h) What determines the size of a PCR product? The positions where the two primers anneal to the template. The PCR product spans from the 5’ end of one primer to the 5’ end of the other primer on the opposite strand. In this experiment: positions 15,971 to 16,411 = 440 bp.
i) What are the two functions of the dNTPs? 1. They serve as the building blocks (monomers) for new DNA strand synthesis — Taq polymerase incorporates them one by one to extend the primer 2. They provide the energy for the phosphodiester bond formation — when a dNTP is incorporated, two phosphate groups (pyrophosphate) are released, providing the energy that drives the reaction
j) Why is MgCl₂ included in the reaction? Mg²⁺ ions are an essential cofactor for Taq DNA polymerase. Magnesium stabilizes the enzyme-substrate complex and facilitates the catalytic mechanism by coordinating with the phosphate groups of the dNTPs. Without Mg²⁺, Taq polymerase has very low or no activity.
DNA Isolation (Chelex method): - Cheek cells collected by swab → dissolved in water → centrifuged to pellet cells → supernatant discarded - Cell pellet resuspended in water → 40 µl transferred to Tube 1 - Chelex resin added → Chelex binds metal ions that would degrade DNA - Boiled 10 min → lyses cells (releases DNA) + inactivates degradative enzymes - Centrifuged → DNA in supernatant transferred to Tube 2 (the clean template)
PCR Setup: - 20 µl of DreamTaq Master Mix (contains Taq, dNTPs, MgCl₂, buffer, primers) added to PCR tube - 5 µl of template DNA (Tube 2) added → total volume = 25 µl - Placed in thermal cycler → 30 cycles run automatically
A student runs the PCR product on an agarose gel with a 100 bp DNA ladder in a neighboring lane. Under UV light (after ethidium bromide staining), a band at approximately 440 bp confirms the correct PCR product was amplified. If no band appears or the band is the wrong size, the PCR failed or produced a non-specific product.
Agarose gel (DNA): - Separation based on size (length in base pairs) — smaller fragments pass through pores faster and migrate farther - DNA has intrinsic negative charge (phosphate backbone) → migrates toward positive electrode WITHOUT any denaturing agent needed - Run in TAE buffer at neutral pH
Protein gel (SDS-PAGE, E11): - Proteins have variable charges (positive, negative, neutral amino acids) so SDS (sodium dodecyl sulfate) must be added to coat all proteins with uniform negative charge - Separation is then by size (molecular weight), not native charge - Run under denaturing conditions; proteins are unfolded
Key difference: DNA separates by size without any special treatment because it’s uniformly negatively charged. Proteins need SDS to be separated by size.
The phosphate groups (PO₄²⁻) on the sugar-phosphate backbone. Every nucleotide in DNA has a phosphate group that carries a negative charge at physiological pH. Since there is one phosphate per nucleotide, larger DNA fragments carry proportionally more negative charge — but because charge and mass scale together, all DNA fragments migrate based on size, not total charge.
a) What affects migration rate of DNA in agarose gel electrophoresis? - Fragment size (bp) — smaller migrates faster - Agarose concentration — higher % gel = smaller pores = slower migration for all, better separation of small fragments - Voltage applied — higher voltage = faster migration - DNA conformation — linear, circular, supercoiled DNA of the same size migrate differently (linear is standard for PCR products)
b) What chemical stains DNA in this experiment? How? Ethidium bromide (EB) — a fluorescent intercalating dye. It inserts (intercalates) between base pairs in the DNA double helix. When exposed to UV light, EB fluoresces bright orange, revealing where DNA bands are located on the gel.
c) Purpose of including a marker lane in the gel? The 100 bp ladder contains DNA fragments of known sizes (100, 200, 300, 400, 500, 600 bp etc.). By running it alongside your PCR product and comparing where your band migrates relative to the ladder bands, you can estimate the size of your PCR product. It’s the reference ruler of the gel.
d) How does the size of linear DNA relate to migration rate? Inversely proportional — larger DNA = slower migration = closer to the wells. Smaller DNA = faster migration = farther from wells (closer to the positive electrode). On a semi-log plot, fragment size vs. distance migrated gives a straight line.
e) What two blue dyes are included in the marker? Why? Cresol red and orange G (or bromophenol blue and xylene cyanol, depending on the kit). These dyes are included because they migrate at approximately known sizes and are visible to the naked eye — they let you monitor electrophoresis progress in real time without UV light, so you know when to stop running the gel before the DNA runs off the end.
Denaturation (in relation to DNA): The process of separating the two strands of the DNA double helix by breaking the hydrogen bonds between complementary base pairs. In PCR, this is achieved by heating to 94°C. The result is two single-stranded DNA templates.
Annealing (in relation to DNA): The process by which two complementary single-stranded DNA sequences come together and form hydrogen bonds to create double-stranded DNA. In PCR annealing, the primers bind to their complementary sequences on the single-stranded template. The bonds formed are non-covalent hydrogen bonds between complementary base pairs (A-T and G-C).
How to predict PCR product size from the genome map: The map shows: Left primer at position 15,971; Right primer at position 16,411 Size = 16,411 − 15,971 = 440 bp
Why use a 100 bp ladder instead of 500 bp? The PCR product is 440 bp. A 100 bp ladder has rungs at 100, 200, 300, 400, 500, 600 bp — the 400 bp band is just below and 500 bp just above the expected product. This gives precise reference points close to 440 bp for accurate size estimation. A 500 bp ladder has rungs too far apart (500 bp intervals) making it difficult to accurately read a 440 bp band.
How many molecules after 30 cycles from one template? Each cycle doubles: 2³⁰ = 1,073,741,824 (≈ 1 billion copies)
Do you expect your PCR product sequence to vary from classmates? Yes and no. The SIZE of the PCR product will be the same for everyone (all students amplify the same 440 bp region using the same primers). However, the sequence within that region may differ due to SNPs in the hypervariable control region. Most people have a unique SNP pattern in their mtDNA control region, meaning the sequence of the 440 bp band will be slightly different from person to person.
Answer each question. Check answers and rationale in Part 6.
Q1. What is the purpose of heating to 94°C in PCR?
Q2. Why is Taq polymerase used in PCR instead of a normal human DNA polymerase?
Q3. What two essential components must be added to the DreamTaq Master Mix to set up a PCR reaction?
Q4. True or False: The two primers in a PCR reaction should be complementary to each other.
Q5. After 30 PCR cycles starting from a single DNA template molecule, approximately how many copies are produced?
Q6. Which of the following is TRUE about mitochondrial DNA?
Q7. During PCR annealing, what type of chemical bonds form between the primer and the template DNA?
Q8. What are the TWO functions of dNTPs in a PCR reaction?
Q9. Why is MgCl₂ included in the PCR reaction mixture?
Q10. What is the basis of DNA separation in agarose gel electrophoresis?
The basis of DNA separation in agarose gel electrophoresis is SHAPE !!!! circular DNA will always migrate faster than linear !!!
Q11. What chemical is used to visualize DNA bands in the gel under UV light?
Q12. Why is a 100 bp DNA ladder used instead of a 500 bp ladder in this experiment?
Q13. A student forgot to add primers to their PCR reaction. What result would they observe on the gel?
Q14. What is the direction of new DNA strand synthesis by Taq polymerase?
DNA synthesis always proceeds 5’→3’. Taq polymerase (like all DNA polymerases) can only add nucleotides to the free 3’-OH end of a growing strand. It reads the template 3’→5’ and builds the new strand 5’→3’.
Q15. You amplified a 440 bp segment of your mtDNA. A classmate also amplified the same region. What would you expect when comparing gel results vs. sequencing results?
| # | Answer | Rationale |
|---|---|---|
| 1 | C | 94°C breaks the hydrogen bonds holding the two DNA strands together, separating dsDNA into two single-stranded templates. This step is called denaturation. Primers cannot bind at this high temperature. |
| 2 | B | Taq polymerase is thermostable — it maintains its structure and function even after repeated exposure to 94°C. Normal human polymerases would denature (unfold and stop working) at this temperature, making PCR impossible. |
| 3 | C | The Master Mix already contains Taq polymerase, dNTPs, MgCl₂, and buffer. The two components you add are the DNA template (your specific sample to be copied) and the primers (to define what region gets amplified). |
| 4 | B | FALSE. If primers were complementary to each other, they’d bind each other instead of the template, forming “primer dimers.” The two primers must each bind opposite strands at opposite ends of the target region, with their 3’ ends pointing toward each other. |
| 5 | D | PCR amplification is exponential: 2³⁰ = 1,073,741,824 ≈ 1 billion copies. Linear amplification (×30) would only give 30 copies. The exponential nature is what makes PCR so powerful. |
| 6 | C | Each cell has hundreds to thousands of mitochondria, each with multiple copies of mtDNA. This means thousands of copies of each mt gene per cell, compared to only 2 copies of each nuclear gene. This is why mtDNA is so easy to amplify from small samples. |
| 7 | C | Primer-template binding is mediated by hydrogen bonds between complementary base pairs (A-T and G-C). These are the same bonds that hold the two strands of the double helix together. They are non-covalent, which is why they can be broken by heating (denaturation) and re-formed by cooling (annealing). |
| 8 | B | dNTPs serve dual roles: (1) as the monomer building blocks incorporated into the new DNA chain by Taq polymerase, and (2) each incorporation releases pyrophosphate (two phosphate groups), providing the chemical energy that drives the reaction forward. |
| 9 | B | Mg²⁺ ions are an essential cofactor for Taq polymerase. They stabilize the enzyme-substrate complex and coordinate with phosphate groups during catalysis. Without Mg²⁺, Taq polymerase has minimal to no activity. |
| 10 | B | DNA separates by size. The agarose gel forms a porous matrix; smaller fragments pass through the pores more easily and migrate farther from the wells. All DNA is uniformly negatively charged, so charge doesn’t vary between fragments — size is the only variable. |
| 11 | D | Ethidium bromide (EB) intercalates between base pairs in double-stranded DNA. When exposed to UV light, EB fluoresces, making the DNA bands visible as bright bands on the gel. Cresol red is a tracking dye used to monitor gel running progress. |
| 12 | B | The PCR product is ~440 bp. The 100 bp ladder has bands at 100, 200, 300, 400, 500 bp — the 400 bp band is just below and 500 bp band just above the expected product, allowing precise interpolation. A 500 bp ladder jumps in 500 bp increments, making it impossible to accurately estimate a 440 bp band. |
| 13 | C | Without primers, Taq polymerase has no 3’-OH end to extend from — DNA polymerase cannot initiate synthesis de novo. No primers = no synthesis = no PCR product = no band on the gel. |
| 14 | B | DNA synthesis always proceeds 5’→3’. Taq polymerase (like all DNA polymerases) can only add nucleotides to the free 3’-OH end of a growing strand. It reads the template 3’→5’ and builds the new strand 5’→3’. |
| 15 | C | All students use the SAME primers amplifying the SAME region → all PCR products are the same size (440 bp) → same position band on gel. HOWEVER, the mtDNA control region is hypervariable — most people have unique SNP patterns. So while the bands look the same size on a gel, sequencing would reveal individual differences in the actual nucleotide sequence. |
No answer choices — write your answers from memory!
FRQ 1. PCR stands for: polymerase chain reaction
What is the overall purpose of PCR? amplify segments of DNA, make copies of segments of DNA !!! _______________________________________________________________
FRQ 2. List ALL the essential components needed in a PCR reaction tube and what each one does:
| Component | Function |
|---|---|
| 2 primers | primers will tell the substrate (taq polyemarase) WHERE to begin extending the DNA template, flanks the template !!! |
| DNA template | need a template in order to make copies |
| dntps (A, G, T, C) | nucleotide base pairs that are complementary to the DNA that will extend and make copies of the DNA |
| taq polymerase | stabilizes the DNA, thermostable, survives high heat environments |
| cofactors + buffer | mgcl2, buffer gives the mixture ideal pH environment |
FRQ 3. Fill in the three PCR steps:
| Step | Temperature | Duration | What happens to DNA |
|---|---|---|---|
| 1. ____denaturation_________ | ___94____°C | 30 sec | double stranded DNA separates into single stranded DNA, hydrogen bonds break |
| 2. _____annealing________ | ____58___°C | 30 sec | cooling down the DNA allows the primers to begin annealing the template DNA |
| 3. ____EXTENSION ___ | ____77 ___°C | 30 sec | allows the DNA to copy and extend, taq polymerase extends HERE. |
FRQ 4. Why is Taq polymerase used instead of a human DNA polymerase? Where does it come from?
Taq polymerase is used because it is thermostable and survives high heat environments, whereas DNA polymerase denatures at high temperatures. it comes from a hot spring.
FRQ 5. What is the limitation of Taq polymerase compared to human DNA polymerase?
the limitation of taq polymerase is that it has no proof reading abilities, leading to more mutations when compared to DNA polymerase.
FRQ 6. After 30 cycles of PCR starting from 1 template molecule, how many copies are produced? Show your reasoning:
1 billion, 2^30, exponential copies
FRQ 7. The two primers in a PCR reaction are complementary to each other. True or False? Explain why:
false, they should NOT be complementary to each other, if they were complementary to each other they would bind to one another. they are non-complementary therefore they will bind to opposite ends of the dna strand and extend from the 3-OH portion .
FRQ 8. What determines the SIZE of a PCR product?
the primers, where the primers are synthesized to bind to .
?
In this experiment, the left primer is at position 15,971 and the right primer is at position 16,411. What is the expected size of the PCR product? ___440____ bp
FRQ 9. What are the TWO functions performed by boiling cells in the DNA isolation step?
FRQ 9. 1. Lyses (breaks open) the cells to release the DNA from inside 2. Inactivates cellular proteins — including nucleases (which degrade DNA) and proteases — that would interfere with PCR
lyses the cell to release the DNA from inside
inactivates cellular proteins (including nucleases) (which degrade DNA) and proteases- that would interfere with PCR
FRQ 10. What does Chelex resin do in the DNA isolation procedure? Why is this important for PCR?
FRQ 10. Chelex resin is a chelating agent that binds and sequesters metal ions (such as Mg²⁺, Ca²⁺, Fe³⁺) from the sample. These metal ions catalyze the degradation (breaking) of DNA. Without Chelex, trace metals in the crude cell lysate would destroy the template DNA before PCR could amplify it. Chelex protects the DNA, ensuring PCR-ready template.
FRQ 11. Explain how DNA separates during gel electrophoresis. Include: what charge DNA has, which direction it migrates, and what property determines how far it migrates.
DNA separates by size and charge. DNA is negative, therefore it will travel towards the positive electrode (anode), size- smaller fragments will migrate faster
FRQ 12. What is ethidium bromide and how does it allow visualization of DNA?
EB:
dye
insert itself between the base pairs of dsDNA
flouresces under UV light
only lights up WHEN bound to DNA
FRQ 12. Ethidium bromide (EB) is a fluorescent chemical dye that intercalates (inserts) between the base pairs of double-stranded DNA. Once intercalated, it fluoresces bright orange/yellow when exposed to UV light. Because EB only fluoresces strongly when bound to DNA, wherever DNA bands have migrated on the gel will appear as bright glowing bands under UV, allowing visualization.
FRQ 13. What is the purpose of the DNA ladder (size marker) in gel electrophoresis?
to meausre the size of the DNA.
Why is a 100 bp ladder more appropriate than a 500 bp ladder for this experiment?
because the 100bp ladder increases in increments of 100, allowing for a more precise measurement of the DNA, that is estimated to have a size of 440bp. the 500 bp ladder will increase in increments of 500, so it will be too big of a ladder for a precise measurement.
FRQ 14. List THREE characteristics of mitochondrial DNA that make it useful for this type of PCR experiment:
FRQ 14. Any three of: 1. mtDNA is present in thousands of copies per cell (hundreds of mitochondria × multiple genome copies each) → easy to amplify even from small samples 2. mtDNA is found in the mitochondria of virtually all cells including cheek epithelial cells 3. The control region is hypervariable — it accumulates SNPs at ~10× the rate of nuclear DNA, making it useful for studying individual variation 4. The entire mt genome sequence is known (sequenced in 1981) 5. mtDNA can be amplified from old, degraded, or tiny samples due to the high copy number
------------------------------------------------------------------------???
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FRQ 15. Two students in the same lab run the same PCR reaction amplifying the same 440 bp mtDNA region. Would you expect their gel bands to be in the same position? Would you expect their sequences to be identical? Explain both answers:
the size would be expected to be the same, however, the sequence would not be identical because of SNPs. this accounts for diversity and evolution.
FRQ 16. What is a SNP? What makes the mtDNA control region especially rich in SNPs?
** REVIEW !!!
FRQ 16. A SNP (Single Nucleotide Polymorphism) is a variation at a single nucleotide position in the DNA sequence between individuals. For example, one person might have an A at position 16,223 while another has a G.
The mtDNA control region is especially rich in SNPs because it is a noncoding region — mutations there do not affect any protein sequence and therefore are not selected against during evolution. As a result, point mutations accumulate freely at approximately 10 times the rate of nuclear DNA, creating high variability between individuals.
FRQ 17. A student's gel shows NO band in their lane, but the positive control (provided by TA) shows a clear 440 bp band. List THREE possible reasons for the student's missing band:
FRQ 17. Any three of: 1. Forgot to add template DNA → nothing to amplify 2. Forgot to add primers → Taq has no starting point, no synthesis 3. Failed DNA isolation — no DNA was extracted (boiling step missed, centrifuge error) 4. Non-functional Taq polymerase (degraded or not added) 5. Thermal cycler malfunction — cycles didn’t run at correct temperatures 6. Forgot to add dNTPs → no building blocks for synthesis 7. Pipetting error — added wrong volumes or contaminated the tube
errors in the heating or cooling
no primers added
no DNA template?
FRQ 1. PCR = Polymerase Chain Reaction Purpose: To exponentially amplify a specific target segment of DNA so that billions of copies are produced from even a tiny or degraded starting sample.
FRQ 2.
| Component | Function |
|---|---|
| Template DNA | The original DNA molecule to be copied |
| Primers (×2) | Short single-stranded sequences that flank the target region and give Taq polymerase a starting point |
| Taq polymerase | Heat-stable enzyme that synthesizes new DNA strand from the primer |
| dNTPs (A, T, G, C) | Building blocks for the new DNA strand; also provide energy for bond formation |
| MgCl₂ | Required cofactor for Taq polymerase activity |
| Buffer | Maintains proper pH and ionic conditions for the reaction |
FRQ 3.
| Step | Temperature | Duration | What happens to DNA |
|---|---|---|---|
| 1. Denaturation | 94°C | 30 sec | Hydrogen bonds break; dsDNA separates into 2 single strands |
| 2. Annealing | 58°C | 30 sec | Primers bind to complementary sequences on each single strand via hydrogen bonds |
| 3. Extension | 72°C | 30 sec | Taq polymerase extends primer in 5’→3’ direction, synthesizing a new complementary strand |
FRQ 4. Taq polymerase is thermostable — it can survive and function after repeated exposure to 94°C, which is required for the denaturation step. Normal human DNA polymerases would unfold and permanently lose activity at this temperature, making repeated PCR cycles impossible. Taq polymerase comes from Thermus aquaticus, a bacterium naturally found in hot springs at ~72–75°C in Yellowstone National Park.
FRQ 5. Taq polymerase lacks a proofreading (3’→5’ exonuclease) function, resulting in a higher mutation rate (~1 error per 10,000 bases incorporated). Human DNA polymerase has proofreading that catches and corrects errors. Newer thermostable polymerases (like Pfu) have proofreading and are used when accuracy is critical.
FRQ 6. PCR is exponential: each cycle doubles the number of molecules. After 30 cycles: 2³⁰ = 1,073,741,824 ≈ 1 billion copies from a single template molecule.
FRQ 7. FALSE. The two primers are NOT complementary to each other. If they were, they would bind each other in solution (forming “primer dimers”) instead of binding the template. The two primers are each complementary to OPPOSITE strands at OPPOSITE ends of the target region, with their 3’ ends pointing toward each other so that Taq can extend toward and through the target region.
FRQ 8. The size of the PCR product is determined by the positions where the two primers anneal on the template. The PCR product spans from the beginning of one primer to the beginning of the other primer on the opposite strand. Size = 16,411 − 15,971 = 440 bp
FRQ 9. 1. Lyses (breaks open) the cells to release the DNA from inside 2. Inactivates cellular proteins — including nucleases (which degrade DNA) and proteases — that would interfere with PCR
FRQ 10. Chelex resin is a chelating agent that binds and sequesters metal ions (such as Mg²⁺, Ca²⁺, Fe³⁺) from the sample. These metal ions catalyze the degradation (breaking) of DNA. Without Chelex, trace metals in the crude cell lysate would destroy the template DNA before PCR could amplify it. Chelex protects the DNA, ensuring PCR-ready template.
FRQ 11. DNA carries a negative charge due to the phosphate groups (PO₄²⁻) on its sugar-phosphate backbone. When placed in an electric field, negatively charged DNA migrates toward the positive electrode (anode). The property that determines how far a fragment migrates is its SIZE (in base pairs). Smaller fragments pass more easily through the pores of the agarose gel and therefore migrate faster and farther from the wells. Larger fragments move more slowly and remain closer to the wells.
FRQ 12. Ethidium bromide (EB) is a fluorescent chemical dye that intercalates (inserts) between the base pairs of double-stranded DNA. Once intercalated, it fluoresces bright orange/yellow when exposed to UV light. Because EB only fluoresces strongly when bound to DNA, wherever DNA bands have migrated on the gel will appear as bright glowing bands under UV, allowing visualization.
FRQ 13. The DNA ladder contains DNA fragments of known sizes (e.g., 100, 200, 300, 400, 500, 600 bp). By comparing how far your PCR product migrated relative to these known bands, you can estimate the size of your product.
A 100 bp ladder is more appropriate because the PCR product is ~440 bp. The 100 bp ladder has bands at 400 bp and 500 bp — close reference points that allow accurate interpolation of a 440 bp band. A 500 bp ladder has intervals too large (500 bp apart) to precisely estimate a 440 bp product.
FRQ 14. Any three of: 1. mtDNA is present in thousands of copies per cell (hundreds of mitochondria × multiple genome copies each) → easy to amplify even from small samples 2. mtDNA is found in the mitochondria of virtually all cells including cheek epithelial cells 3. The control region is hypervariable — it accumulates SNPs at ~10× the rate of nuclear DNA, making it useful for studying individual variation 4. The entire mt genome sequence is known (sequenced in 1981) 5. mtDNA can be amplified from old, degraded, or tiny samples due to the high copy number
FRQ 15. Gel bands: YES — both bands would appear at the same position (~440 bp). All students use the same primers amplifying the same region, so the PCR product is always the same length regardless of individual SNPs. Sequences: NOT necessarily identical. The mtDNA control region is hypervariable and most people carry a unique pattern of SNPs. So the 440 bp band contains the same length of DNA but potentially different nucleotide sequences between students.
FRQ 16. A SNP (Single Nucleotide Polymorphism) is a variation at a single nucleotide position in the DNA sequence between individuals. For example, one person might have an A at position 16,223 while another has a G.
The mtDNA control region is especially rich in SNPs because it is a noncoding region — mutations there do not affect any protein sequence and therefore are not selected against during evolution. As a result, point mutations accumulate freely at approximately 10 times the rate of nuclear DNA, creating high variability between individuals.
FRQ 17. Any three of: 1. Forgot to add template DNA → nothing to amplify 2. Forgot to add primers → Taq has no starting point, no synthesis 3. Failed DNA isolation — no DNA was extracted (boiling step missed, centrifuge error) 4. Non-functional Taq polymerase (degraded or not added) 5. Thermal cycler malfunction — cycles didn’t run at correct temperatures 6. Forgot to add dNTPs → no building blocks for synthesis 7. Pipetting error — added wrong volumes or contaminated the tube