BME100 s2016:Group11 W1030AM L4

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Contents

OUR TEAM

Name: Kyle Durrant
Name: Kyle Durrant
Name: Brandon Taylor
Name: Brandon Taylor
Name: Eric Wright
Name: Eric Wright
Name: studentRole(s)
Name: student
Role(s)
Name: studentRole(s)
Name: student
Role(s)
Name: studentRole(s)
Name: student
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
  • DNA/primer mix, 8 tubes, 50 μL 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 tip only once. Never re-use disposable pipette tips or samples will be cross contaminated
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine: shared by two groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G11 + Positive control 87806
G11 - Negative control 99993
G11 1-1 Patient 1, replicate 1
G11 1-2 Patient 1, replicate 2
G11 1-3 Patient 1, replicate 3
G11 2-1 Patient 2, replicate 1
G11 2-2 Patient 2, replicate 2
G11 2-3 Patient 2, replicate 3


DNA Sample Set-up Procedure

  1. Step 1 : Collect a DNA sample from the patient
  2. Step 2 : Move DNA into PCR tube
  3. Step 3 : Add the first primer to the PCR tube containing the extracted DNA
  4. Step 4 : Add the second primer to the PCR tube containing the extracted DNA
  5. Step 5 : Add nucleotides to the PCR tube (Adenine, thymine, cytosine and guanine)
  6. Step 6 : Add DNA Polymerase to the PCR tube
  7. Step 7 : Finally, place the PCR tube into the DNA Thermal Cycler


OpenPCR program

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 2 minutes

FINAL STEP: 72°C for 2 minutes

FINAL HOLD: 4°C





Research and Development

PCR - The Underlying Technology

The template DNA's function is to serve as a template in order for an enzyme, polymerase, to create a complementary strand based on the template DNA. Due to this, one double stranded DNA molecule is converted into two. Both of these are identical to the first. The primers are short pieces of DNA with any sequence of nucleotides that serves as a starting point for DNA synthesis. In the case of PCR, the primers are custom designed in a laboratory to be around 20 nucleotides long and will target one place in the genome. The Taq polymerase is a form of DNA polymerase that is from a strain of Thermus aquaticus bacteria which can survive in near boiling temperatures. This is important because the DNA polymerase in our bodies would break down prior to reaching the 95 degrees Celsius threshold in a polymerase chain reaction. This Taq polymerase's function is to copy a cell's DNA before it divides in two. The function of the deoxyribonucleotides are to serve as the building blocks that the DNA molecules themselves are made up of. By adding a mixture of four types of deoxyribonucleotides (Adenine's, Cytosine's, Guanine's, and Thymine's) into the PCR reaction, the DNA polymerase is able to grab these nucleotides and attach them to the end of the primers.

Thermal Cell Cycling is broken down into 6 steps: Initial Step, Denature, Anneal, Extend, Final Step, and Final Hold. During the Initial Step, the components are heated to 95°C for 3 minutes. During this process, the hydrogen bonds holding together the double helix structure of the Template DNA break from the high heat, causing the strands to separate. After the Initial Step, the Denature step follows, keeping the components heated at 95°C for an additional 30 seconds. At this time, all of the Template DNA is separated into single strands. Next, the Anneal phase calls for a rapid cool down to 57°C for 30 seconds. During this cool down, the single-stranded DNA naturally wants to pair back together. Before the strands can pair back together, the Primers lock onto target sequences on the single strands, preventing other strands from joining together. After the short Anneal phase, the solution is heated to 72°C for 30 seconds in the Extend phase. During this phase, the Template DNA serves as the anchor for primers and DNA Polymerase to work off of. The Primers remain attached at their target sequences, and serve as a target for Taq Polymerase to latch onto. Taq Polymerase locates the primer and begins adding complementary nucleotides to the strand. These complementary nucleotides, otherwise known as Deoxyribonucleotides (dNTP’s), are the building blocks of the new strand of DNA. The Final Step of Thermal Cell Cycling occurs for 3 minutes after the Extend period and remains at a constant temperature of 72°C. During this phase the Taq Polymerase continues the dNTP’s to the Template DNA until finished. The processes before this point are repeated a desired amount of time to produce more and more copies of the targeted DNA. After the desired time has elapsed, the Final Hold stage brings the solution and all of the components in it down to 4°C. This cool temperature cools the replicated DNA to prevent degradation and effectively stops the Polymerase Chain Reaction from occurring.


Base Pairing

The nucleotides in DNA pair their bases according to hydrogen bonding. Adenine and thymine always pair together and cytosine and guanine always pair together.

DNA base pairing occurs during the extend phase and the final step. In the extend phase the primer is already placed and polymerase will attach at 72 degrees celsius. Once polymerase is attached it will begin pairing nucleotides and will continue to pair them through the final step.



SNP Information & Primer Design

Background: About the Disease SNP

The SNP stands for a single nucleotide polymorphism. The gene that this disease SNP is associated with is B3GNT3 and this is a species that is specifically found in Homo Sapiens. This chromosome is located on 19:17811986, and it was found that the disease that is linked to this specific SNP happens to be non-Hodgkin lymphoma. This SNP is due to a variation in the sequence of DNA among individuals. This is how they obtain SNP's such as the disease non-Hodgkin lymphoma.

Primer Design and Testing

Non-disease forward primer (20 nt): 5’- G T G C G G G C T C C A T C G C A A C G

The numerical position exactly 200 bases to the right of the disease SNP is: 17812186

Non-disease reverse primer (20 nt): 5’- G G A G G A A G G T G T C G C C C C T T

Non-disease forward primer (20 nt): 5’- G T G C G G G C T C C A T C G C A A C G Non-disease reverse primer (20 nt): 5’- G G A G G A A G G T G T C G C C C C T T

This was first done by designing a non-disease forward primer based on the position of the SNP. The numerical position was then found by looking at the 200 bases to the right of the disease SNP. From this, the Non-disease reverse primer was able to be found. Finally, from this data the disease forward primer and the disease reverse primer were able to be obtained. When the Non-disease reverse primer is tested, it provides a 220 bp sequence from the chromosome. However, the disease specific primer from before provides no matches when tested.

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