BME100 f2017:Group2 W1030 L4

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OUR TEAM

Name: Kaitlyn Nielsen
Name: Alexis Crego
Name: Krysten Walker
Name: Will Goodall
Name: Ryan Shapiro

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: 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-contaiminated
  • Cup for discarded tips
  • micropipettor
  • OpenPCR machine: shared by 2 groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G2 + Positive control none
G2 - Negative control none
G2 1-1 Patient 1, replicate 1 34668
G2 1-2 Patient 1, replicate 2 34668
G2 1-3 Patient 1, replicate 3 34668
G2 2-1 Patient 2, replicate 1 59805
G2 2-2 Patient 2, replicate 2 59805
G2 2-3 Patient 2, replicate 3 59805


DNA Sample Set-up Procedure

  1. Step 1
  2. Step 2
  3. Step 3...


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






Research and Development

PCR - The Underlying Technology
Description of image

Description of image

Source: PCR http://learn.genetics.utah.edu/content/labs/pcr/ (accessed Oct 18, 2017).



Functions of the Components

Template DNA is the original DNA used to begin the replication process. The function of the primer is to attach to the base of the targeted DNA and form nucleotides. TAQ polymerase copies the DNA of the targeted area prior to the denaturing of the DNA. Deoxyribonucleotides are the building blocks that DNA molecules are made of.

Steps of Thermal Cycling

The initial step in the PCR process is raising the temperature of the sample to 95 degrees Celsius, which is almost boiling point, for two minutes. This causes the double helix to separate into two separate DNA strands. The temperature is then lowered to 50 degrees Celsius to allow primers to attach to the base of the targeted strands. Then the temperature is raised to 72 degrees Celsius and the DNA polymerase is activated and locates a primer. After 30 seconds, nucleotides begin to form and extend to replicate another strand of DNA identical to the original. Finally, the temperature is lowered to 4 degrees Celsius to prevent further replication, and is a suitable temperature for storage.

DNA Base Pairing

There are four types of molecules called nucleotides, A, T, C, and G, that pair up to create DNA. The base Adenine (A) anneals with the base Thymine (T), and base Guanine (G) anneals with base Cytosine (C).

Thermal Cycling

Base-pairing occurs during annealation and extension. The reason being that annealing requires primers to pair up with the single DNA strands, and they can only be compatible if the bases properly pair up with the correct nucleotides. Extension is when the primers start building identical DNA onto another strand to bond with the original one, which also requires the proper set of nucleotides in order to be built correctly and bond together.


SNP Information & Primer Design

Background: About the Disease SNP SNP is a single-nucleotide polymorphism that occurs throughout everyone's DNA. SNP's can replace nucleotides ( adenine, thymine, cytosine, and guanine) with a different type of a nucleotide resulting in the person having more than one allele. A nucleotide is a nitrogenous base, s-carb sugar phosphate group and is the building blocks of nucleic acids. The position of the disease is 44907853 and when it is diseased it appears as 44908053. A healthy allele in SNP goes from CTG to CCG when diseased. As you can see for an allele to be diseased a small change occurs which shows that just the smallest error can have serious effects like mutations and diseases.


Primer Design and Testing This image shows the forward and reverse primer of PCR and the effect temperature has on it. The primers are synthesized by an enzyme and allow DNA polymerase to attach the new nucleotides and repair the strand. This occurs at 72.7 degrees for the forward sequence, and 65.3 degrees for the reverse sequence. The bond can break and allow DNA polymerase to repair the diseased codon. The forward primer is at the beginning of the gene sequence and the reverse is at the end. The reverse sequence is suppose to compliment the forward strand.

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