BME100 f2017:Group12 W0800 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: Amy Nystrom
Roles- Data Recorder, Report Manager
Name: Kira Stryker
Role(s)-Sample watcher/label maker
Name: Priscilla Han
Role(s)- Micropipetter
Name: Vishnu Karthigeyan
Role(s)-Sample Watcher/label maker
Name: Jason
Role(s)- Time Manager

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat and disposable gloves
  • PCR reaction mix (contains Taq DNA polymerase, MgCl2, and dNTP’s), 8 tubes, 50 μL each:
  • DNA/primer mix (Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer), 8 tubes, 50 μL each
  • A strip of empty PCR tubes
  • Disposable pipette tips
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine

PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G12 + Positive control none
G12 - Negative control none
G12 1-1 Patient 1, replicate 1 76650
G12 1-2 Patient 1, replicate 2 76650
G12 1-3 Patient 1, replicate 3 76650
G12 2-1 Patient 2, replicate 1 80605
G12 2-2 Patient 2, replicate 2 80605
G12 2-3 Patient 2, replicate 3 80605


DNA Sample Set-up Procedure

  1. Add extracted DNA to PCR tube
  2. Add Primer 1 to PCR tube - it will attach to the first site
  3. Add Primer 2 to PCR tube - it will attach to the second site
  4. Add nucleotides (building blocks of DNA) to PCR tube
  5. Add DNA Taq Polymerase to PCR tube
  6. Place PCR tube in Thermocycler
  7. Repeat the above steps for each sample of DNA required
  8. Set the program for the Thermocycler and initiate it

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 30 seconds

FINAL STEP: 72°C for 2 minutes
FINAL HOLD: 4°C

PCR, short for Polymerase Chain Reaction, is a method used by scientists to replicate and amplify the specific region of DNA that is of interest. PCR is done by adding primers to the DNA and repeatedly heating and cooling the mixture which has the effect of simulating the temperatures that are necessary for DNA replication to occur. The first step is for the temperature to go up to 95 degrees Celsius for 30 seconds so that the DNA “denatures,” or splits from its double-stranded form into single strands. After this step, the temperature goes back down to 57 degrees Celsius for 30 seconds which allows the primers to bind to the strands. Then the temperature goes back up slightly to 72 degrees Celsius for 30 seconds so that the enzyme DNA Taq polymerase can attach to the primed sequences and add nucleotides. This is the first cycle and the PCR will happen for 25 cycles in total.





Research and Development

PCR - The Underlying Technology


Steps:

PCRRG121.png
1. At 95°C, DNA denatures from its double helix form into 2 single strands.

PCRRG122.png
2. At 52°C, the primers attach to their complementary DNA sequences at the beginning of the target sequence.

PCRRG123.png
3. Once the mixture is reheated to 72°C, the DNA taq polymerases attach to the primers.

PCRRG124.png
4. DNA taq polymerase synthesizes new strands of DNA by using free floating nucleotides nearby and the template DNA.

PCRRG125.png
5. Once the DNA taq polymerase finishes, a new strand of the target sequence is formed.

PCRRG126.png
6. Steps 2 through 5 are repeated to produce ample copies of the original target sequence. This process continues until the temperature stops changes or the supply of nucleotides runs out.



Defining the components and their functions:
- DNA is short for deoxyribonucleic acid. It is the genetic instructions for development, functioning and reproduction of humans.
- Template DNA is the original strand of DNA that is about to be separated and copied.
- Primers attach to each end of the DNA segment that is going to be copied; they will only bind the specific segment.
- Nucleotides are the pieces that make up nucleic acids. They letters that represent the four different kinds are A,T,C,G.
- DNA Polymerase read the nucleotides on the DNA and matches them with its corresponding nucleotides. The one being used in the experiment has been specially designed to withstand high temperatures.
- DNA Thermal Cycler heats and cools the samples to exact temperatures at set times.

Base pairing sets: A matches with T, T matches with A, C matches with G and G matches with C.

SNP Information & Primer Design

Background: About the Disease SNP

The human genome is made of many long strings of code composed of nucleotides in a specific order, that instruct the cell which proteins to produce. Single nucleotide polymorphisms, or SNPs (which are pronounces like the word "snips"), are a result of an accidental alteration in a single component or nucleotide in the DNA, which can lead to a variation in the proteins produced by the cell. When a piece of DNA is altered in such a way that it causes a change in the proteins produced, that leads to a change in the function of the cell, as proteins are the mechanisms through which the cell works. So when a SNP produces this type of change, it can lead to disease in the cell, and potentially fatal problems in the organism. Due to the fact that these morphisms in DNA can lead to such drastic outcomes in the organism, it is very useful for medical professionals to be able to identify the location of a SNP, so that they have a better understanding of how to approach removal of the disease SNP.

Primer Design and Testing

(Question 15 on worksheet)

In the primer design, we seek to find the forward and reverse primer and read them from 5’ to 3’. We want to find the base pair in the genome that contains the error. This base pair and the 19 base pairs before it form the the forward primer that has a total of 20 base pairs. In this case, the primer started at 44907853 and it was 5’-​ ​​AGCGGCCAGCGCTGGGAACC. The reverse primer is 200 base pairs prior to the base pair that had the mutation and it serves as a reference point during PCR. Thus, we can find the primer position to be 44908053 by adding 200 from the base pair position of the forward prime and the reverse primer to be 5’-​ ​​ CAGGCCCCCCAAGACTTAGC. We test these primers by inputting them into the genome site and we found the healthy 220-base pair long primer. The results can be found below. When the disease mutation was inputted, we didn’t find the primers since you wouldn’t expect to find it in a healthy human’s genome.

PCR SS.png