BME100 f2016:Group8 W8AM L4

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
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]=OUR TEAM=

Name: Dhantin Kumar
Name: Juan Pablo Robayo
Name: Blake Sentyrz
Name: Andre Bordeleau
Name: Jasmine Davis

LAB 4 WRITE-UP

Protocol

Materials

  • Micropipettor
  • Disposable Pipette tips (never to reuse)
  • Lab Coat/Disposable Gloves
  • PCR Reaction Mix (8 tubes, 50 μL in each, contains Taq DNA polymerase, MgCL2, and dNTP's).
  • DNA/Primer mix (8 tubes, 50 μL in each, each contains a different template DNA. All tubes have the same forward primer and reverse primer).
  • Strip of empty PCR tubes
  • Cup for discarded tips
  • OpenPCR machine


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G# + Positive control none
G# - Negative control none
G# 1-1 Patient 1, replicate 1 45539
G# 1-2 Patient 1, replicate 2 45539
G# 1-3 Patient 1, replicate 3 45539
G# 2-1 Patient 2, replicate 1 33494
G# 2-2 Patient 2, replicate 2 33494
G# 2-3 Patient 2, replicate 3 33494


DNA Sample Set-up Procedure

  1. The DNA samples from our patients are extracted and transferred to the correctly labeled PCR tubes (as shown above).
  2. Pipette 50µL of the DNA primers to the DNA samples in each of the PCR tubes. Discard pipette.
  3. 50µL of the PCR reaction mix (DNA polymerase and nucleotides) is added to the samples which are labeled accordingly.
  4. The samples are then added to the PCR machine and run with the various temperatures and times as listed below.

OpenPCR program

Step Details
HEATED LID: 100°C
INITIAL STEP: 95°C for 2 minutes
NUMBER OF CYCLES: 25, Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and extend at 72°C for 30 seconds
FINAL STEP: 72°C for 2 minutes
FINAL HOLD: 4°C



PCR-process.gif




Research and Development

PCR - The Underlying Technology

Q1. What is the function of each component of a PCR reaction?

Component Function
Template DNA: The Template DNA contains the target site that we wish to examine. Upon making multiple copies over several cycles, the Template DNA will yield over a billion copies of our target site by the 30th cycle.
Primers: These attach onto the Template DNA when it 'unzips' and stretches out. The isolated target sites we seek will be tagged with these primers.
Taq Polymerase: The polymerase attaches to the primers and runs down the length of unfurled DNA, reading the nucleotides and attaching their corresponding matches as it goes along. As our cycles progress, the Taq Polymerase will be attaching to the abundant target sites as well as the longer strands of DNA. The target DNA will easily become more abundant than any other DNA strand in the solution.
Deoxyribonucleotides (dNTP's): Loose nucleotides float around in the solution, ready to be taken by the Taq Polymerase to be strung into a corresponding sequence for the other DNA strand.


Q2. What happens to the components (listed above) during each step of thermal cycling?

Steps Temperature and Time Function
INITIAL STEP 95°C for 3 minutes The DNA strands are heated to near boiling and the two strands straighten out
Denature​ 95°C for 30 seconds The DNA strands separate completely
Anneal 57°C for 30 seconds Primers bind to the target DNA sequence (SNP) and bracket the region of DNA that is to be copied
Extend 72°C for 30 seconds Taq Polymerase binds to the end of the primer site and matches complementary nucleotides to the template DNA and extends that strand
FINAL STEP 72°C for 3 minutes The Taq polymerase has fully extended the DNA strands from the primer site to form new double-helix strands of the target DNA
FINAL HOLD 4°C For short-term storage of the newly amplified DNA strands


Q3. DNA is made up of four types of molecules called nucleotides, designated as A, T, C and G. Base-pairing, driven by hydrogen bonding, allows base pairs to stick together. Which base anneals to each base listed below? If you need help, use the “Build a DNA Molecule” tool at

http://learn.genetics.utah.edu/content/begin/dna/builddna/

Base Base Pair
Adenine (A) Thymine (T)
Thymine (T) Adenine (A)
Cytosine (C) Guanine (G)
Guanine (G) Cytosine (C)


Q4. During which two steps of thermal cycling does base-pairing occur? Explain your answers.

Base-pairing occurs between the Annealing and Extension steps. After the two DNA strands have denatured into single strands, the samples are cooled down in the PCR machine to 57°C. In this step, the short DNA primers bind to the target SNP in the newly created single-stranded DNA (the nucleotide sequence that is being amplified). Then the temperature is raised slightly to 72°C so that the Taq Polymerase binds to the end of the primer site, and it now extends the primers down the DNA strand and matches the base pairs with their counterparts (as shown in question 3) to form new nucleotide strands of the desired target SNP of the DNA.


Components and their functions in PCR reactions

Each PCR reaction has four main components. The first component is a template DNA which contains the target site that is wished to be examine. Upon making multiple copies over several cycles, the Template DNA will yield over a billion copies of the target site by the 30th cycle. Second are the primers which attach onto the Template DNA when it unzips and stretches out. The isolated target sites sought for will be tagged with these primers. Third is the taq polymerase which, attaches to the primers and runs down the length of unfurled DNA, reading the nucleotides and attaching their corresponding matches as it goes along. As our cycles progress, the Taq Polymerase will be attaching to the abundant target sites as well as the longer strands of DNA. The target DNA will easily become more abundant than any other DNA strand in the solution. The final component are the Deoxyribonucleotides (dNTP's) which float around in the solution, ready to be taken by the Taq Polymerase to be strung into a corresponding sequence for the other DNA strand.

Steps of thermal cycling


In the initial step of thermal cycling the DNA is heated to 95°C for 3.5 minutes in order to straighten denture and separated the DNA helix into two single strands. During Anneal, the solution is held at 57°C for 30 seconds and primers bind to the target DNA sequence (SNP) and bracket the region of DNA that is to be copied. Then during the extension period the solution is heald at 72°C for 30 seconds and the Taq Polymerase binds to the end of the primer site and matches complementary nucleotides to the template DNA and extends that strand. For the final step the solution is heald at 72°C for 3 minutes and the Taq polymerase will fully extended the DNA strands from the primer site to form new double-helix strands of the target DNA. The solution is then held at 4°C for short-term storage of the newly amplified DNA strands.

Base pairs

Each nucleotide base has its own pair (Adenine with Thymine, Thymine with Adenine, Cytosine with Guanine and Guanine with Cytosine). These pairs make up DNA bonds (with the addition of hydrogen bonding) and coding.


When base pairing occurs

Base-pairing occurs between the Annealing and Extension steps. After the two DNA strands have denatured into single strands, the samples are cooled down in the PCR machine to 57°C. In this step, the short DNA primers bind to the target SNP in the newly created single-stranded DNA (the nucleotide sequence that is being amplified). Then the temperature is raised slightly to 72°C so that the Taq Polymerase binds to the end of the primer site, and it now extends the primers down the DNA strand and matches the base pairs with their counterparts (as shown in question 3) to form new nucleotide strands of the desired target SNP of the DNA.





SNP Information & Primer Design

Background: About the Disease SNP
In SNP, the nucleotides form the basic structure to create nucleic acids in DNA, and polymorphism is when a population has genetic variation within and allows for natural selection to occur. This variation of SNP of is found in the species "Homo Sapiens", located on the chromosome "4:113367751". The Clinical Significance for this variation is listed as "Pathogenic". Based off the studies of the "Proceedings of the National Academy of Science of the United States of America", SNP is linked to the condition known as "Cardiac Arrhythmia Syndrome". ANK2 is the term used for identifying a gene which is followed immediately by and ID and stands for Ankyrin 2, neuronal. It is responsible for encoding members of the ankyrin family of proteins that are in charge of linking the integral membrane proteins to the spectrin-actin cytoskeleton. Alleles are single or multiple forms of a gene that appear due to undergoing mutation and is found in the same area on a chromosome. The allele associated with the disease has the codon "ATC". The numerical position of the SNP is located at "113367751".
Primer Design and Testing
"Non-disease Primers"
Non-disease forward primer of 20nt: 5' - GGA CAG CTC AGC AAC AGC ACT - 3'.
Non-disease reverse primer of 20nt: 5' - TAA AAA GTA TTT AAA AAC TA - 3'.
The numerical position that is 200 bases right of the disease SNP is "113367951". Our primers work since the result was a 220 bp long sequence of the chromosome in part 4.
"Disease Primers"
Disease-forward Primer of 20nt: 5' - GGA CAG CTC AGC AAC AGC AAT - 3'.
Disease-reverse Primer of 20nt: 5' - TAA AAA GTA TTT AAA AAC TA - 3'. NO matches were found