DNA (PTC) lab report Materials and Methods draft

PTC introduction

Bitter gourds are notoriously bitter fruits that are eaten like vegetables. (“Bitter gourd” (Links to an external site.) by Abhijeet Kamble; MGB CEE, Wikimedia Commons (Links to an external site.) is licensed under CC BY-SA 4.0) (Links to an external site.)
It’s a matter of taste
The way we perceive flavors, whether it’s pancakes, tacos, or savory pile of noodles, is a combination of smell and taste. For the taste component of flavor, our brains rely on signals from specific taste receptors found on the tongue. These receptors detect chemicals in our food. But without the receptor to detect specific chemicals, there is no signal to tell your brain whether or not that chemical is present.
Many factors contribute to how individuals experience taste. One of those is genetics, which determines the phenotype of your tastebuds and therefore what chemicals you can detect and how sensitive you are to them. Once such gene is the PTC gene (discovered only in 2003!), which codes for a protein that is a receptor for detecting phenylthiourea-phenylthiocarbamide (PTC).
In the human population, there are several different versions, or alleles of the PTC gene, only two of which are very common. So one person could have two of the same allele OR two different alleles.
Why does each person have two alleles for each autosomal (non-sex chromosome) gene (not just PTC)?
Read this article (Links to an external site.) from the Genetic Science Learning Center for more information on PTC and its biological implications.
Why might the ability to taste PTC, a synthetic molecule not found in nature, be related to evolutionary advantage?
How is it thought that PTC tasting is inherited?

DNA extraction
In order to analyze DNA, first you need to get DNA. While DNA is found in all living cells, so is a lot of other material! That material (organelles, polysaccharides, lipids, proteins, etc.), both within and between cells can interfere with downstream work (like PCR!) that a researcher would want to do with DNA.
So, the first step for doing our PTC genetic analysis is to extract and purify DNA for testing.
DNA extraction protocols vary immensely, mostly depending on what kind of cells you are using. But, in order to extract DNA you need to do three basic steps:
1. Collect cells
2. Lyse cells
3. Isolate DNA
1. Collect Cells
A common and minimally invasive way to collect human cells is from the inside of the cheek. These epithelial cells are easily and painlessly removed.
Here is how we do it specifically for the PTC lab:
1. Swirl saline in your mouth to collect cells from your cheeks
2. Centrifuge your sample to pull the heavy materials (cells) to the bottom of the tube, forming a pellet.
3. Remove the supernatant (liquid above the pellet), leaving only the cells

Now, we’re ready to work with these cells!
2. Lyse Cells
You have cells, but now you need to break them open and get the DNA out from the inside. Lysis can be achieved in several different ways, and we are going to do it by boiling the cells, causing them to rupture due to the high temperature.

For animal cells like the whole cheek cell above, how membranes do you need to break through in order to get the DNA?

In animal cells, you need to break open the plasma membranes and nuclear membranes. What else would you need to break open for plant, fungal, or bacterial cells?
Chelex protects your DNA
Cells have lots of components in addition to DNA. One of the things found in cells is DNAse, an enzyme that degrades DNA. Normally, your DNA is safely in the nucleus and away from the DNAse in cells. But when you start breaking up cells in order to get that DNA out, everything gets combined and the DNA is vulnerable to digestion.

This is where chelex comes in. Chelex binds up free magnesium ions. DNAse needs Mg2+ as a cofactor in order to function. Without free magnesium, DNAse gets inactivated and your precious DNA is safe!

3. Isolate DNA
Now you have a crude lysate, which contains all the debris from the rest of the cells, along with the DNA you want. That debris is larger and more dense than the DNA, so you can centrifuge it down to form a pellet.
After you put this crude lysate in the centrifuge, what fraction (supernatant or pellet) should you keep?

That’s right! Your debris is in the pellet, and your less dense DNA is now in the supernatant. Just transfer that supernatant to a new tube, and you’ll be ready for PCR!

Some key points:
• Chelex beads are used to protect DNA from breaking down when the cell solution is being heated.
• Lysis of the cells is done by boiling the solution to release the DNA from the cell.
• Centrifugation separates the larger cell debris and chelex beads from the rest of the DNA in solution.
See how it’s done
Watch this video for a rundown of the lab protocol to extract DNA for PCR:

Experiment 8 part B: Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR)
PCR is a powerful technology that does something simple: copy DNA. The primers used determines what particular span of DNA gets copied. All cells need to replicate their DNA in order to divide, and by learning about that process we have developed the ability to copy specific pieces of DNA (like the PTC gene!) for diagnostic, research, and much more!
Watch this video for a review of how PCR works:

What “ingredients” do you need to do PCR?
1. PCR primers are short pieces of single-stranded DNA, usually around 2020 nucleotides in length. Two primers are used in each PCR reaction, and they are designed so that they flank the DNA sequence of interest. For instance, we are using a primer that matches a sequence upstream (on the 5′ side) of the PTC gene and a primer that matches a sequence downstream (on the 3′ side) of the PTC gene. The primers are given sequences that will make them bind to opposite strands of the template DNA, just at the edges of the region to be copied. The primers bind to the template by complementary base pairing as illustrated below.

2. Taq polymerase is the enzyme that synthesizes new DNA using dNTPs (dATP, dCTP, dGTP, and dTTP). All cells need DNA polymerase in order to replicate. Taq is the specific DNA polymerase originally isolated from the bacterium Thermus aquaticus.
3. dNTPs are the deoxynucleotides that Taq polymerase uses make new copies of DNA. They are the monomer building blocks that are added to growing strands of DNA, or polymers of nucleotides.

4. Template DNA is needed, because DNA polymerase adds bases to a strand according to the complementary base on the other, antiparallel strand. Without a template, DNA polymerase has nothing to copy!
Procedure
1. Obtain a PCR tube containing Ready-to-Go PCR beads. Ready-to-Go PCR beads contain everything needed for PCR (including dNTP and Taq polymerase) but NOT water, primers, or template DNA.
2. Add 22.5 μl primers and loading dye mix. The purpose of the loading dye is for ease of loading onto the agarose gel later for gel electrophoresis.
3. Add 2.5 μl Daisy’s DNA. Make sure you add directly into the 22.5 μl primers/loading dye mix. Not add to the wall of the PCR tube.
4. Mix well and centrifuge briefly to bring down any droplets remaining on the side of the PCR tube.
5. Store your sample on ice until you are ready to set up the Mastercycler for PCR.
6. Place your PCR tube in the Mastercycler with programming described below
+ DNA + Taq polymerase + dNTP + Primers ⟹

Program steps to PCR amplify PTC
PCR is accomplished by going through a sequence of from denaturing, annealing, and extension steps 30 times, or cycles.
Step 1: DNA denaturing. DNA template (DNA extracted from Daisy’s cheek cells, week 10 material) at 98∘C for 30 sec.
Step 2: Primer annealing at 55∘C for 45 sec.
Step 3: Extension at 72∘C for 45 sec.
Final step: cool down to 4∘C until ready for storage.
See how it’s done
Last week, we successfully extracted DNA from Daisy’s cheek cells. This week, we are going to use that DNA for PCR.
Watch this video for a rundown of the lab protocol to do PCR on your epithelial cell DNA, as demonstrated by Daisy and Prof. Elizabeth Taylor:

How to cite your sources
Citations allow us an organized way to acknowledge our sources AND to give people who read our work the chance to go back and investigate that source themselves.
In-text Citation
When you are writing and sharing information that you have found elsewhere (rather than, say reporting the results of your own experiment), it’s important to include an in-text citation. This is a bit like a quick footnote so people can find the relevant citation in your list of references at the end of your work. Typically in-text citations include the author and date of the work you are citing:
Viruses are typically described as being very small, typically between 20nm and 400nm in diameter (Gutierrez, 2017). However, the recent discovery of giant viruses larger than some bacteria are challenging the idea of viruses as small biological particles (Wu and Johnson, 2018).
References List
Look here (Links to an external site.) for detailed explanations of how to write citations for a references page in APA format.
I’ve included some excerpts below of some common types of references, with their APA formatted citation.
Scholarly Journal Article
Note that if you access a journal article online, it should be cited as a scholarly article, NOT a website.
Conger, Rand. 1997. “The Effects of Positive Feedback on Direction and Amount of Verbalization in a Social Setting.” American Journal of Sociology 79:1179-259.
Coe, Deborah L., and James D. Davidson. 2011. “The Origins of Legacy Admissions: A Sociological Explanation.” Review of Religious Research 52(3):233-47.
Phillips, Reginald M., and S. H. Bonsteel. 2010. “The Faculty and Information Specialist Partnership Stimulating Student Interest and Experiential Learning.” Nurse Educator, 35(3), 136-138. doi: 10.1097/NNE.0b013e3181d95090.
Lab manual
Department of Biology. (2018). BIOL 101 lab manual. Chapel Hill, North Carolina: University of North Carolina at Chapel Hill.
Website
Purdue University. 2012. “Purdue University’s Foundations of Excellence Final Report: A Roadmap for Excellent Beginnings.” Retrieved Nov. 21, 2014 (http://docs.lib.purdue.edu/provost_pubs/1/).

Restriction Digestion
Genetic Variation
We are using restriction digestion in order to detect genetic variation in the human PTC gene. While there are other methods one could use, restriction digestion is simple and inexpensive, while something like sequencing would take longer and be more expensive.
As a species, Homo sapiens is relatively young. This means that genetically, we haven’t had a very long time to evolve many genetic differences. On average, two humans are only 0.1% different! Compare a human to a chimp, and we’re only about 1.2% different. That means when we sequence a human genome, the vast majority of that sequence is exactly the same among all people on Earth.
The remaining genetic differences we do have can inform us about our biology. The differences from person to person in their genomes(all the DNA in your cells) are called variants. Variants are also called polymorphisms, because they are places that the genome is different, or has “more than one shape”.
A single nucleotide polymorphism (SNP) is the smallest type of polymorphism, consisting of just one base pair. Small differences like this are the basis for many kinds of tests, such as paternity, diagnosing some genetic diseases, and forensic identification. See below for an example of a SNP, highlighted in red. The only difference between the gene sequences is a single nucleotide base pair.

Restriction Digestion
Just like animal cells, bacterial cells can get infected by viruses. Restriction enzymes are made by bacteria as part of their immune defenses. Each restriction enzyme binds and then cuts a very specific sequence of double-stranded DNA, which matches a viral genome sequence but not the bacteria’s own DNA. In this way, a bacterial cell can “restrict” a viral infection. The place a restriction enzyme cuts is called a restriction site. If just one base in the restriction site is changed, the restriction enzyme will no longer recognize it and therefore will not cut. See below for an illustration of how a restriction enzyme cuts.

Blunt vs. sticky ends
Restriction enzymes can make two kinds of cuts. Some enzymes cut to produce blunt ends, where the two DNA strands are cut at the same spot. Other enzymes cut to produce sticky ends, where both DNA strands are cut, but the cut on one strand is a few bases away from the cut on the other strand. This leaves a small overhang on either side. Since the bases are exposed along the overhang, they can form hydrogen bonds, making them “sticky” to other complementary DNA sequences. See below for an illustration of these two types of restriction enzyme cuts.

HaeIII: One enzyme in the arsenal
Restriction enzymes are named based on the organism where they were discovered. So HaeIII was isolated from the bacterium Haemophilus aegypticus. We have chosen this enzyme because it happens to recognize a sequence (GGCC) that overlaps the SNP in the human PTC gene TAS2R38. HaeIII cuts between the G and C nucleotides.
What type of end does this leave?

Every enzyme has optimal conditions (e.g. – pH, salinity, temperature, etc.) that, if met, will allow the enzyme to work as fast as possible. The mixture of HaeIII enzyme and PCR products are heated because enzymes work more efficiently at 37C.
You can find out more about HaeIII at this product page (Links to an external site.).
HaeIII distinguishes between PTC taster and PTC non-taster alleles
The PTC taster allele for TAS2R38 has the sequence GCAGGCAGGCCCATT. Will HaeIII cut this DNA sequence? See the figure below for an illustration of what happens!

The PTC non-taster allele for TAS2R38 has the sequence GCAGGCAGACCCATT. Will HaeIII cut this DNA sequence? See the figure below for an illustration of what happens!

This is all clear-cut if you happen to be homozygous for the TAS2R38 gene, but what if you are heterozygous and have one of each allele? Then, the half of your DNA (taster alleles) will be cut and half of your DNA (non-taster alleles) will NOT be cut by HaeIII.

Restriction enzyme review
Watch this video for an overview of how restriction enzymes work, using EcoR1 as an example.

Restriction enzymes are multipurpose tools. For our lab on PTC genotyping, we are using restriction enzymes to determine what PTC allele(s) each person has.
Check out this interactive lab from LabXchange (Links to an external site.) to find out more about how restriction enzymes are used to identify single nucleotide polymorphisms (SNPs).
See how it’s done!
Last week, we successfully PCR-amplified the PTC gene from Daisy’s cheek cell DNA.
Watch this video for a walkthrough of the lab protocol to do a restriction digest of your epithelial cell DNA:

HaeIII SNP Summary
• A single nucleotide polymorphism (SNP) is the change of a single base pair in a gene sequence.
• Restriction digestion is when DNA is cut at a specific site recognized by a restriction enzyme.
• The HaeIII enzyme and the PCR products are heated so HaeIII can work more efficiently.
• The Haelll enzyme recognizes, attaches to, and cuts the PTC taster SNP sequence.
• Nontasters have a SNP that the Haelll enzyme does not recognize and is not cut into smaller DNA fragments.