practicum 3

Question 1

What does PCR stand for?

Answer 1

Polymerase Chain Reaction – a method to amplify a specific DNA sequence.

Question 2

What are the 3 repeated steps in PCR and what happens in each?

Answer 2

• Denature (95°C): DNA strands separate.
• Anneal (50–65°C): Primers bind to target DNA sequence.
  -Extend (72°C): Taq polymerase adds nucleotides to synthesize new DNA strands.

Question 3

What is the source of genomic DNA in the PTC experiment?

Answer 3

Cells collected from the inside of your cheek (buccal cells).

Question 4

What general class of receptor is responsible for PTC tasting?

Answer 4

G-protein-coupled receptors (GPCRs) – involved in taste signal transduction

Question 5

What is an SNP?

Answer 5

Single Nucleotide Polymorphism – a variation at a single base pair in DNA that can lead to different traits.

Question 6

What protein does the TAS2R38 gene encode?

Answer 6

A bitter taste receptor protein involved in detecting the PTC compound.

Question 7

What are the two phenotypes associated with TAS2R38?

Answer 7

• Taster – perceives PTC as bitter.
• Non-taster – does not perceive bitterness from PTC.

Question 8

What are the two alleles of TAS2R38?

Answer 8

• PAV: Associated with tasting PTC.
• AVI: Associated with non-tasting PTC.

Question 9

What is Fnu4H1 and its role?

Answer 9

• A restriction enzyme that cuts DNA at a specific site present only in one of the alleles (PAV or AVI).
• Significance: Used to determine which allele is present by whether or not the PCR product is cut.

Question 10

How are the PCR products analyzed?

Answer 10

Via gel electrophoresis after digestion with Fnu4H1.
You determine which allele(s) you have based on the pattern of bands:

Cut = One allele
Uncut = Other allele

Question 11

Purpose of this lab

Answer 11

• Extract DNA
• Amplify the TAS2R38 gene via PCR
• Identify your genotype for the PTC-tasting trait using restriction digestion and gel electrophoresis

Question 12

PCR Purpose

Answer 12

Amplify specific DNA sequences quickly and precisely.

Question 13

What problems does PCR solve?

Answer 13

Like transformations, PCR enables the study of specific genes.
PCR solves:

• Low DNA quantity by amplifying it.
• Allows genotype identification without cloning into a plasmid.

Question 14

PCR Reagents

Answer 14

• Template DNA: The DNA sample to be amplified (cheek cells).
• Primers: Short DNA sequences that flank the target gene and start replication.
• dNTPs (A, T, C, G): Building blocks of new DNA.
  -Taq DNA polymerase: A heat-stable enzyme that synthesizes DNA.

Question 15

Source of Template DNA in PTC lab

Answer 15

Cheek cells (buccal epithelial cells).

Question 16

How were PCR products analyzed?

Answer 16

• Digested with Fnu4H1.
• Run on an agarose gel.
• Visualized under UV light to see which bands are present.

Question 17

Genotype-PAV/PAV (TT), Taster

Answer 17

Gel Pattern: Digested bands only (short fragments)

Question 18

Genotype-AVI/AVI (tt), Non-taster

Answer 18

Undigested band only (long fragment)

Question 19

PAV/AVI (Tt), Medium

Answer 19

Both long and short bands present

Question 20

Genotype

Answer 20

The actual genetic makeup (e.g., PAV/PAV)

Question 21

Phenotype

Answer 21

The observable trait (e.g., taster)

Question 22

Steps of the Experiment Explained

Answer 22

• PCR Amplification
  Target a region of TAS2R38 gene.
• Fnu4H1 Digestion
  Cuts DNA if SNP is present in PAV allele.
• Gel Electrophoresis
  Separate fragments by size.
  Determine genotype by band pattern.