BISC110/F12: Lab 6 Taster SNP1

From OpenWetWare

Revision as of 16:54, 13 June 2012 by Michelle L. Labonte (Talk | contribs)
Jump to: navigation, search
Wellesley College

BISC110/112- Introduction to Cell Biology Lab- Fall 2012





Since Fox's serendipitous discovery, variation in the ability to taste PTC (and other related bitter compounds such as those found in broccoli!) has become one of the most studied of human genetic traits. On the surface, PTC sensitivity appears to be inherited as a simple Mendelian trait with two alleles, T for taster and t for nontaster; in reality its inheritance is much more complicated. Today we know that PTC sensitivity is mediated by the PTC gene (aka TAS2R38) that encodes a bitter taste receptor (a heteromeric G-protein coupled receptor to be exact) which is found on the surface of cells of the tongue. Kim et. al. (2003), published a ground-breaking paper entitled, Positional Cloning of the Human Quantitative Trait Locus Underlying Taste Sensitivity to Phenylthiocarbamide, in the journal Science, which can be downloaded in PDF form through the Wellesley College Library's on-line subscription to Science. To learn even more about the PTC gene, check out the review article by Kim and Drayna (2006) and another review by S. Wooding (2006). In class we will view the video “The Science of Picky Eaters”.

Additional studies of the PTC gene in humans across the globe have revealed that there are two common alleles, taster and nontaster; however, there are at least nineteen other rare alleles that affect the taster phenotype. These different forms of the gene code for proteins that vary in their ability to bind bitter compounds such as PTC. The two common alleles of the PTC gene differ by three single nucleotide polymorphisms (SNPs) which are shown below. SNPs (pronounced “snips”) represent the simplest type of genetic variation between individuals. A SNP refers to a specific location in the genome where different people have been shown to have a different nucleotide. If more than one percent of the population has a different base at a particular location, that location is considered a SNP. If less than one percent of the population has a different base, it is considered a mutation.

Polymorphic Sites in the PTC Taster Gene
Nucleotide position Taster Nontaster
145 CCA
785 GCT
886 GTC

The common nontaster allele (or haplotype) has a G at nucleotide position 145 (G145), T at position 785 (T785) and A at position 886 (A886). This nontaster allele produces a polypeptide with alanine, valine and isoleucine at these sites and therefore is referred to as the AVI allele. The common taster allele C145, C785, and G886, produces a polypeptide with proline, alanine, and valine at these sites, and is referred to as the PAV allele.

The SNP at position 785 is useful for determining your PTC genotype since the common taster sequence in this region forms a Fnu4H1 restriction site. In contrast, replacing C785 with T785 in the common nontaster allele eliminates this restriction site. (A restriction site is a specific DNA sequence that is recognized and cleaved by a specific restriction enzyme or endonuclease.) There are two other Fnu4H1 restriction sites within the PTC gene, but we will use primers specifically designed to amplify the region flanking the one polymorphic Fnu4H1 site while excluding the other two. Using these primers in a polymerase chain reaction (PCR) generates a 303 base pair (bp) fragment. Restriction digestion of this PCR product with Fnu4H1 will yield one 303 bp fragment for the common nontaster allele and two shorter fragments (65 bp and 239 bp) for the common taster allele. Visit the Dolan DNA center web site at [1] to learn more about PCR and see an animation of how it works.

Fnu4H1 Restriction Site
N can be any of the 4 bases


Figure 1. Sequence of the common Taster allele of the PTC gene. The 3 SNPs are highlighted in blue with the nucleotide found in the common nontaster allele shown below the taster. The position of the forward and reverse PCR primers are shown in green and red, respectively. The Fnu4H1 site you will exploit for genotyping is underlined in black at position 785.

In two-weeks of lab experiments and a third lab meeting of discussion, you will be provided with a phenotype and determine the genotype at the PTC locus of a hypothetical Wellesley student. You will also explore some of the ethical issues surrounding personal genomics and learn more about writing a scientific paper. In the first week you will be assigned a PTC phenotype. You will also begin the process of determining your assigned PTC genotype by extracting and purifying human PTC DNA contained within bacterial plasmids (a plasmid is a circular piece of DNA). You will then use the polymerase chain reaction (PCR) to amplify a region of DNA that contains a SNP in the PTC gene. PCR is a technique which exploits in vitro DNA replication by a heat stable DNA polymerase to produce millions of copies of a specific DNA sequence. In addition, we will watch a video about some of the ethical issues related to the use of SNPs and other genetic information. In the second lab you will determine your assigned PTC genotype by digesting your PCR product with the restriction enzyme Fnu4H1 and analyzing the products by agarose gel electrophoresis. You will analyze the course data and make figures in preparation for a data analysis meeting. You will also outline your introduction and discussion sections to use in an in-lab workshop to prepare you for writing a scientific paper on your findings.

Lab 6 - Taster SNP Week 1

You will use a kit to isolate bacterial plasmid DNA (containing part of the human PTC gene). You will chemically break these cells open and use a silica-based spin column to separate the DNA from the other cellular components.

  1. Your instructor will assign you a number and a TASTER phenotype defined as either T (taster), NT (non-taster), or PT (partial taster). Record your assigned number and phenotype in your lab notebook so you don't forget them! You will need to remember your number so you can record your genotype results next to the phenotype results you have just obtained.
  2. You will be provided with a bacterial cell pellet in a 1.5-mL microfuge tube. Be sure that this tube has the same number that you were just assigned.
  8. Apply the supernatent from the step 6 to your labeled QIAprep spin column by decanting.



PART III: PCR of your PTC gene (using PuRe Taq Ready-To-Go Master Mix beads (GE Healthcare)
Next, you will set up a polymerase chain reaction (PCR) designed to amplify from your extracted DNA the region of the PTC gene which contains the SNP at position 785 (the one which creates a Fnu4H1 site if a C is present). We need to use PCR so we can specifically analyze the region of the PTC gene surrounding the SNP at position 785. The forward and reverse priming sites for our reactions are shown in Figure 1. In your notebook record the sequences of the forward and reverse primers. Use the convention of writing the primer sequence beginning with the 5’ end. Have your instructor check your sequences before proceeding.


Here is the cycle we will use for your samples:
This is followed by 40 cycles with the following pattern:
1 minute at 95C, for “melting” the DNA
1 minute at 72C, for synthesizing new DNA

PART IV. SNPs and bioethics




The Answers to the following questions will be turned in at the beginning of Lab 7. You may download a .doc file of these questions here Media:Taster_questions110112_F12.doc‎ to use as a template for your answers.

The following terms should be used to fill in the blanks for questions 1-3. Each term must be used one time.


1. Madeline was in the lab when her friend opened up a container of PTC. All of a sudden she noticed a horrible taste in her mouth. At this point, we know that Madeline has a taster _______________ but we are not yet sure of her__________________.

2. The PTC gene on one of Katherine’s chromosomes was examined at it contains a C at position 145, a C at position 785, and a G at position 886. Katherine has the common nontaster PAV _______________ or _________________.

3. One of Leticia’s copies of chromosome 14 was examined. Like 12% of human chromosomes examined, she has a T at position 883 of gene X. Like 0.2 % of human chromosomes examined, she has a G at position 664 of gene Y. Her T at position 883 of gene X is considered a _____________ and her G at position 664 of gene Y is considered a ____________.

4. Ingrid has the following SNPs on one copy of chromosome 20:

G at position 334 of gene A
T at position 586 of gene A
C at position 23 of gene B
A at position 437 of gene B
A at position 978 of gene B


______________________________ G334, T586

______________________________ C23, A437, A978

______________________________ G334, T586, C23, A437, A978

5. Think about the results you expect from next week's electrophoretic separation. After considering all you now know about PTC gene polymorphisms, draw the expected migration locations of the Fnu4H1 digested PCR product obtained from a homozygous taster C785/C785(left lane), a homozygous nontaster T785/T785 (center lane) and a heterozygote C785/T785(right lane) on a diagram of an agarose gel (template shown below).


Personal tools