What are the main steps of a thermal cycling profile, and how can these steps be adjusted to optimize, or troubleshoot, a reaction that may not be yielding positive results?
 


BIOL3251 GENETICS LAB FALL 2018

ASSIGNMENT 4 Name:__________________________ PCR: Polymerase Chain Reaction Lab section:_____________________

The polymerase chain reaction, or PCR, allows us to make many copies of targeted regions of the genome for a number of different downstream applications. In this project, you are PCR-amplifying your mitochondrial HVR1 region so that you can obtain the DNA sequence for that region. The ultimate goal is to determine your haplotype or haplogroup based on that region, which will provide you with insight into your deep ancestry.

Consider the class discussions you had on PCR this week. Use this information to answer the following questions:

1. What are the primary components of a typical PCR reaction, and what does each of these components do?

2. How can these components be adjusted to optimize, or troubleshoot, a reaction that may not be yielding positive results?

3. What are the main steps of a thermal cycling profile, and how can these steps be adjusted to optimize, or troubleshoot, a reaction that may not be yielding positive results?

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BIOL3251 GENETICS LAB FALL 2018

The entire human mitochondrial genome is just over 16,500 base pairs (bp) in length, and it is organized as a single, circular chromosome. The Cambridge Reference Sequence, fully annotated and downloaded from GenBank, is provided below. You will use it to complete the follow questions.

4. Using a highlighter, colored pen, or colored pencil, mark and label the entire HVR 1 and HVR 2 regions on the Cambridge Reference Sequence below.

5. On the CRS below, mark and label the priming sites for the left and right primers used in today’s PCR experiment. Use a different color than was used for marking the entire HVR1 and HVR2 regions.
 
 Primer name Primer sequence

mtDNA_PCR_L 5’-tcattggacaagtagcatcc-3’ mtDNA_PCR_R 5’-aggctaagcgttttgagctg-3’
 
 Hints:

1. The primers are designed to amplify HVR1 and HVR2 as one contiguous unit.

2. The right primer is the reverse complement of its intended priming site. 3. The primers were designed to anneal (or bind) just outside of the HVR

regions to ensure that the entire region would be amplified and sequenced. 4. The primers were designed by Dr. Cahoon specifically for this class project,

so Google searching is pointless (just saving you the time and frustration :).

6. What size amplicon (i.e., PCR product) do you expect these primers to generate?