BME100 f2016:Group13 W1030AM L4

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

Name: Collin Friedrich
Role(s)
Name: Jordyn Hartunian
Role(s)
Name: Ricardo Dominguez
Role(s)
Name: Corey Miles
Role(s)
Name: Lauryn Wallace
Role(s)

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50 µL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s

(http://www.promega.com/resources/protocols/product-information-sheets/g/gotaq-colorl ess-master-mix-m714-protocol/)

  • DNA/ primer mix, 8 tubes, 50 µL each: Each mix contains a different template DNA. All tubes

have the same forward primer and reverse primer

  • A strip of empty PCR tubes
  • Disposable pipette tips: only use each only once. Never reuse disposable pipette tips. If you

do, the samples will become cross-contaminated

  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine: shared by two groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G13 + Positive control none
G13 - Negative control none
G13 1-1 Patient 1, replicate 1 33805
G13 1-2 Patient 1, replicate 2 33805
G13 1-3 Patient 1, replicate 3 33805
G13 2-1 Patient 2, replicate 1 18900
G13 2-2 Patient 2, replicate 2 18900
G13 2-3 Patient 2, replicate 3 18900


DNA Sample Set-up Procedure
Step 1: Obtain source of DNA desired for PCR.
Step 2: Insert extracted DNA to the Polymerase Chain Reaction tubes to manipulate temperature.
Step 3: Then add Primer 1 to the PCR tube/s because primers are the tools used to copy DNA sequences.
Step 4: Add Primer 2 to the PCR tube/s which attaches to the second site.
Step 5: Add nucleotides to PCR tube/s which will be used to create DNA copies.
Step 6: Add DNA Polymerase to the PCR tube/s which reads the DNA code and attaches complementary nucleotides.
Step 7: Place the PCR tube/s into the DNA Thermal Cycler and start it.


OpenPCR program

  • Heated LID: 100 degrees Celsius
  • Initial Step: 95 degrees Celsius
  • Number of Cycles: 25
  • Denature at 95 degrees Celsius for 30 seconds,
  • Anneal at 57 degrees Celsius for 30 seconds, and
  • Extend at 72 degrees Celsius for 30 seconds
  • Final Step: 72 degrees Celsius for 2 minutes
  • Final Hold: 4 degrees Celsius

The initial step in the process is denaturing, or the process of separating the DNA into two different strands. This occurs by heating up the sample DNA to 95 degrees Celsius which breaks the DNA into two. Next, the DNA sample is annealed to 57 degrees celsius in order to have the primers attach to complementary matches on one of the two DNA strands previously created during the denaturing process. The sample is then heated up to seventy-two degrees celsius in order to allow the taq polymerase to create nucleotides which then creates two strands allowing for the cycle to occur again. This process is done twenty-five different times so that there are many copies of the DNA sample the experimenter is interested in. Then the DNA sample is set at 72 degrees celsius so that the remaining single strands are extended completely. Finally, the sample is set at 4 degree celsius in order to make sure that taq polymerase does not keep reacting.





Research and Development

PCR - The Underlying Technology


Components of PCR reactions

The purpose of a Polymerase chain reaction is to make copies of DNA. The template DNA is essential because it is the segment of DNA that is being copied. Primers, DNA sequences which in the laboratory, match the segment of DNA that you want to copy. This allows DNA Polymerase to start base pairing at the area of interest. Two primers are necessary for this process since one attaches itself at the beginning of where the DNA will be copied and the other primer attaches itself to where the DNA will cease to be copied. TAQ Polymerase is a type of DNA Polymerase derived from a strain of bacteria that can survive at very high temperatures. Using this particular base pairing tool is important because high temperatures are required to manually seperate DNA strands. Other DNA Polymerases are not ideal because they can not survive at these temperatures. The function of TAQ Polymerase is to attach itself at the primer and add nucleotides until the next primer, essentially copying the DNA. Deoxyribonucleotides, often referred to as dNTP's, are the single units composing DNA. There are four types :adenine, guanine, cytosine, and thymine. These building blocks comprise the template DNA and it's copy.

Thermal Cycling

Thermal cycling consists of six steps beginning with heating the DNA template at 95 °C for 3 minutes in order to denature. The denaturing process which occurs at the same temperature for 30 seconds is comprised of the DNA strands separating from one another. In the annealing step, where the temperature is 57°C for 30 seconds, two primers are attached to the target sites, at the beginning and end of the DNA sequence which you want to copy. In the extension step which occurs at 72°C for 30 seconds, the TAQ polymerase is activated, therefore attaching itself to the first primer. During the following three minutes, the TAQ polymerase adds complimentary deoxyribonucleotides until it reaches the next primer, the end of the sequence desired to be copied. At 4°C, the PCR reaction ceases.

Base Pairing

Driven by hydrogen bonding, the four nucleotides are paired to their complimentary base by DNA polymerase. The base pairs are as follows: Adenine pairs with Thymine, Thymine pairs with Adenine, Guanine pairs with Cytosine, Cytosine pairs with Guanine. This process of base pairing is vital in copying a template strand of DNA.

Base Pairing in the Thermal Cycling Process

Base pairing occurs in the annealing and extension steps. During annealing, the primers attach to the target DNA, acting as base pairs for the starting and ending positions of DNA replication. Base pairing is then carried out in the extension step by TAQ polymerase throughout the template strand, following the primer.

SNP Information & Primer Design

Background: About the Disease SNP

A common SNP disease, known as single-nucleotide polymorphism, is found in homo sapiens. Nucleotides form the basic structural unit of nucleic acids such as DNA, while polymorphism refers to the genetic variation within a population, upon which natural selection can operate. The chromosome variation is located on Chromosome-4, making SNP clinically significant because it is pathogenic. The most common medical condition linked to this disease is Cardiac Arrhythmia Syndrome.

Primer Design and Testing

ANK2 stands for Ankyrin-2 which plays a key role in activities such as cell motility, activation, proliferation, contact, and the maintenance of specialized membrane domains. The disease associated allele contains the CTC-->ATC codon. Alleles have two or more alternate forms of a gene that arise by mutation and are found at the same place on the chromosome. The numerical position of SNP is 113367751. The non-disease forward primer we created came to the following: 5'-GACAGCTCAGCAACAGCAC-3'. The non-disease reverse primer that we created had a numerical position at 113367951, and was 5'-TAAAAAGTATTTAAAAACTA-3'. The pair of disease SNP-specific primers for the Disease Forward Primer was 5'-GACAGCTCAGCAACAGCAA-3' and the Disease Reverse Primer was 5'-TAAAAAGTATTTAAAAACTA-3'. When we searched for the non-disease primer in the forward direction with the SNP-specific primers, there were no matches. This is due to the forward specific primer being the disease-associated allele.


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