hamood

.pdf

School

University of Toronto, Scarborough *

*We aren’t endorsed by this school

Course

A01

Subject

Biology

Date

Oct 30, 2023

Type

pdf

Pages

Uploaded by ChefMolePerson4101

Lab 4 and 5 Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability Overview In Lab 4 and 5 you will be introduced to some techniques (DNA isolation, Polymerase Chain Reaction (PCR), restriction enzyme digests, and gel electrophoresis) that are frequently used in molecular biology. You will use these techniques to predict your own ability to taste bitter compounds. After isolating your own DNA, you will amplify, using PCR, a short (221bp) fragment of each of your chromosome sevens, a portion of the TASR38 locus or the "bitter tasting gene". Due to the fact that variation of a single nucleotide exists in this region of the gene in human populations, you will be able to use a restriction enzyme digest, followed by gel electrophoresis to determine if you are a homozygous taster (TT), a homozygous non-taster (tt), or a heterozygote (Tt) capable of tasting bitter compounds less strongly than the tasters. Background Information Discovery of PTC It was in the early 1930s that Arthur L. Fox would serendipitously discover that taste had a genetic basis. While pouring phenylthiocarbamide (PTC) (a white powder) into a bottle, Fox would expose both himself and a work colleague, C.R. Noller, to its taste. Noller complained of the compound's bitter taste, while Fox insisted he could taste nothing. Ever after directly sampling, the discrepancy between the two men's ability to taste PTC persisted. This initial observation prompted Fox to test a large amount of people, where he would find that distinct variation existed in human populations regardless of age, sex, and ethnicity. His findings were published and garnered the attention of geneticists who were beginning to explore the organization of the human genome. Albert F. Blakeslee, a prominent plant geneticist at the time of Fox's discovery, would replicate Fox's results and show that the inability to taste PTC is a recessive trait that varies in the human population. Genetics of PTC In 2003, the PTC gene (TASR38) was identified and sequenced. This gene is found on chromosome 7 and is 1002 bp in length. It codes for a taste receptor protein on the tongue. Single-nucleotide polymorphisms.

Within the 1002bp of the PTC gene there are three nucleotide positions that vary within the human population. Each position is known as a single nucleotide polymorphism (SNP). One specific combination of these three SNPs, confers the strongest ability to taste bitter compounds. In today's lab you will be amplifying only one of these SNPs, the one that occurs at nucleotide position 145 in the PTC gene (See Figure 4.1). A taster will have a cytosine ("C') in this position, while a non-taster will have a guanine ('G'). When considering the protein, the taster allele will code for the amino acid proline (codon: CCA) at this position, while a non-taster allele will code for alanine (codon: CGA). We are able to exploit SNPs, to determine the genotype of an individual, through the use of restriction enzymes. Derived from bacteria, restriction enzymes will recognize short specific nucleotide sequences in double-stranded DNA and predictably cut the DNA at this site. The restriction enzyme you will use in this lab is known as Haelll. It will recognize the DNA sequence GGCC and cut it between the G and C (See Figure 4.1). Because only the taster alleles possess this sequence, Haelll is only able to cut the DNA of tasters. Without this sequence of DNA, the non-taster DNA will remain uncut (See Figure 4.1).

Image

Image Description: In chromosome 7, a single nucleotide polymorphism (SNP) exists within the 3' UTR of the TAS2R38 gene, a bitter taste receptor gene. The polymorphism, known as tt, involves a single nucleotide change, resulting in the expression of two distinct proteins, which in turn confer the perception of bitterness in different individuals. By performing PCR amplification followed by restriction enzyme digestion, this SNP can be reliably identified in any individual. Here is a summary of the steps involved: 1. Prepare the DNA sample and PCR primers for the amplification. 2. Amplify the target region using the primers and appropriate polymerase. 3. After the amplification, isolate the PCR product, which is a DNA fragment containing the SNP. 4. Next, digest the PCR product with the appropriate restriction enzyme, in this case, the Hha I enzyme. 5. Perform gel electrophoresis to separate the resulting fragments based on their sizes. 6. The resulting DNA fragments are analyzed under a DNA sequencer or a capillary electrophoresis system to identify the SNP. The PCR amplification ensures that only the target DNA region is amplified, increasing the accuracy of the test. The restriction enzyme digestion provides a more robust and reproducible way to detect the SNP. Ultimately, the identification of this SNP can lead to a deeper understanding of how our genetic makeup influences our perception of bitterness, paving the way for the development of new therapies and treatments for bitter taste disorders.</s> Glossary of Terms The following terms will aid you in your understanding of this lab. Please refer to these terms as you prepare for both Lab 4 and Lab 5.

Aliquot: The division of a quantity of material into smaller equal parts. Allele: Different forms of a gene or different sequences of a gene. Annealing: In PCR, this refers to the binding of single-stranded DNA primers to complementary DNA template sequences, once the template DNA has been denatured or is single-stranded. Chelate: To bind metal ions in solution. An example of a common chelating agent is EDTA (ethylene diamine tetra acetic acid) Cofactor: lon or other small molecule needed by an enzyme to function properly. For example, Taq DNA polymerase requires Mg** to function properly. Mg?* is considered a cofactor. Denaturation: In PCR, this refers to the separation of paired complementary DNA strands through the application of heat. Heat will disrupt the hydrogen bonds that exist between the complementary base pairs of double stranded DNA. DNase: Enzyme that degrades DNA. dNTPs: Commonly used abbreviation for all four deoxynucleoside triphosphates (dATP, dTTP, dGTP, and dCT) used in DNA synthesis. Extension: In PCR, the elongation of a primer (once annealing has occurred) by addition of complementary dNTPs by a Taq DNA polymerase. Extension proceeds in a 5' to 3' direction. Genomic DNA: The sum total of DNA that is found within a cell. Genotype:

The genetic makeup of an organism or cell; the particular combination of alleles present in an individual organism. Heterozygous: Genotypes when two alleles of a given gene are different. Homozygous: Genotypes when two alleles of a given gene are the same. Lysis: The process of rupturing a cell to release its contents. Lysis of a cell is required in the process of DNA isolation. Master Mix: A premixed reagent solution designed for PCR reactions, containing all the necessary components (dNTPs, a forward and reverse primer, buffer, and Taq DNA polymerase). Nucleotide: A fundamental unit of DNA or RNA consisting of a sugar (deoxyribose or ribose), phosphate group, and a nitrogenous base (adenine, cytosine, guanine, thymine, or uracil) PCR: Polymerase Chain Reaction. A selective and highly sensitive method for amplifying (making many copies) targeted pieces of DNA, making a functional concentration of this portion of a genome, which can be used for further analysis and/or manipulation. Phenotype: The expression of a physical, behavioural, or biochemical trait (eg. eye colour). Primer: A small chain of nucleotides (usually 16-24 bases long) that provides a free 3' carbon for DNA polymerase to extend from. Primers for PCR are designed (synthesized in a laboratory) to be complementary to specific sequences near the target DNA sequence. In PCR, there are always two primers used to amplify a specific sequence of DNA, a forward/right primer and a reverse/left primer, one for each strand of the DNA. SNP:

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version