What are the primary components of a typical PCR reaction, and what does each of these components do?
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?
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BIOL3251 GENETICS LAB FALL 2018
Homo sapiens mitochondrion, complete genome NCBI Reference Sequence: NC_012920.1 – full details of the sequence submission with all annotations can be obtained from http://www.ncbi.nlm.nih.gov/nuccore/NC_012920.
Human Mitochondrial Sequence and Annotation aka the Cambridge Reference Sequence (CRS)
This sequence was derived from DNA isolated from placental tissue collected from a caucasian living in Great Britain and was first published in 1981.
The mitochondrial haplotype given to this sequence is H1c
ANNOTATION:
Region or Gene Location Gene product HVR2 1 – 569 hypervariable region 2 TRNF 577..647 tRNA phenylalanine RNR1 648..1601 12S ribosomal RNA TRNV 1602..1670 tRNA valine RNR2 1671..3229 mitochondrially encoded 16S RNA TRNL1 3230..3304 tRNA leucine 1 ND1 3307..4262 NADH dehydrogenase 1 TRNI 4263..4331 tRNA isoleucine TRNQ 4329..4400 tRNA glutamine TRNM 4402..4469 tRNA methionine ND2 4470..5511 NADH dehydrogenase 2 TRNW 5512..5579 tRNA tryptophan TRNA 5587..5655 tRNA alanine TRNN 5657..5729 tRNA asparagine TRNC 5761..5826 tRNA cysteine TRNY 5826..5891 tRNA tyrosine COX1 5904..7445 cytochrome c oxidase I TRNS1 7446..7514 tRNA serine 1 TRND 7518..7585 tRNA aspartic acid COX2 7586..8269 cytochrome c oxidase II TRNK 8295..8364 tRNA lysine ATP8 8366..8572 ATP synthase 8 ATP6 8527..9207 ATP synthase 6 COX3 9207..9990 cytochrome c oxidase III TRNG 9991..10058 tRNA glycine ND3 10059..10404 NADH dehydrogenase 3 TRNR 10405..10469 tRNA arginine ND4L 10470..10766 NADH 4L ND4 10760..12137 NADH dehydrogenase 4 TRNH 12138..12206 tRNA histidine TRNS2 12207..12265 tRNA serine 2 TRNL 12266..12336 tRNA leucine 2 ND5 12337..14148 NADH dehydrogenase 5 ND6 14149..14673 NADH dehydrogenase 6 TRNE 14674..14742 tRNA glutamic acid CYTB 14747..15887 cytochrome b TRNT 15888..15953 tRNA threonine TRNP 15956..16023 tRNA proline HVR1 16001 – 16569 hypervariable region 1
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