BME100 f2016:Group9 W8AM 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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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
G9 + Positive control none
G9 - Negative control none
G9 1-1 Patient 1, replicate 1 45117
G9 1-2 Patient 1, replicate 2 45117
G9 1-3 Patient 1, replicate 3 45117
G9 2-1 Patient 2, replicate 1 82337
G9 2-2 Patient 2, replicate 2 82337
G9 2-3 Patient 2, replicate 3 82337


DNA Sample Set-up Procedure

  1. Step 1

Obtain a source of DNA wanted for PCR

  1. Step 2

Transfer the DNA into a special PCR tube to easily manipulate the temperature

  1. Step 3

Add Primer 1 to the special PCR tube because primers are very effective at copying DNA sequences

  1. Step 4

Add Primer 2 which attaches to second site

  1. Step 5

Add nucleotides to the tube to have a foundation to build more nucleotides off of

  1. Step 6

Add DNA polymerase which read the DNA and assign complementary nucleotides

  1. Step 7

Put the PCR tube into the thermocycler and start 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

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

Once the DNA is inside the thermocycler, the thermocycler heats up the DNA to 95 degree Celsius which begins to unwind the DNA double helix into two separate strands.The thermocycler then drops to 50 degrees Celsius and at this temperature natural pairing of the DNA begins but since there are more primers than DNA strands, the primers lock on their target strands before they can naturally pair. The thermocycler then changes to 72 degrees Celsius and at this temperature the DNA polymerase is activated. The DNA polymerase then locates the primers and add the complementary base pairs. Soon the desired fragments will appear, primer one starts it and primer two ends it. The target sequence will soon become the majority. For example, after 30 cycles the process will have made over one billion target fragments.

Table 1: What is the function of each component of a PCR reaction?

Media:Table1*.xlsx

Table 2: What happens to the components (listed above) during each step of thermal cycling?

Media:Table2*.xlsx

Table 3: 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?

Media:Table3*.xlsx

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

The first step when base pairing occurs is when the temperature changes to 50 degrees Celsius, DNA natural pairs but since there are more primers the primers directly attach to the desired fragments and copies the DNA/nucelotides. DNA polymerase is added at 72 degrees Celsius which locates the primers and adds the complementary base pairs. A series of this process will produce a large amount of the desired DNA.





SNP Information & Primer Design

Background: About the Disease SNP

The disease SNP, single nucleotide polymorphism, is when there is a mutation or unexpected variation in a single nucleotide that happens in a specific portion of the genome. This is fairly rare happening at about 1 in every 1000 bases. This small difference can put an individual at a disadvantage because a mutation of a single base can increase the risk for Alzheimers. However, the presence of SNP provides a way to research the human genome by physical mapping which uses these variations as markers. The types of variations due to SNP can help predict how humans are effected by certain chemicals and how humans develop diseases.

Nucleotide - Monomers of nucleic acid and contains a five carbon sugar, a phosphate group, and a nitrogenous base, these make up DNA. Polymorphism - A variation that develops different types of groups or individuals in a population.

What species is this variation found in? Homo sapiens

What chromosome is this variation located on? 4:113367751

What is listed as the Clinical Significance of this SNP? Pathogenic allele

What condition is linked to this SNP? Cardiac Arrhythmia Syndrome

What does ANK2 stand for? Ankyrin-2

What is the function of ANK2? ATPase binding enzyme binding cytoskeletal adaptor activity

What is an allele? An alternative form of a gene located on a specific location on a chromosome.

The disease associated allele contains what codon? ATC

The numerical position is : 113367751

Non disease forward primer(20nt): GGG ACA GCT CAG CAA CAG CA

The numerical position exactly 200 bases to the right of the disease SNP is: 113367951

Non disease reverse primer(20nt): AAA AAG TAT TTA AAA ACT AG

Disease forward primer(20nt):GGG ACA GCT CAG CAA CAG CT Disease reverse primer(20nt): AAA AAG TAT TTA AAA ACT AG


Primer Design and Testing

The results of the primer test revealed that the non disease primers indicated a 220bp sequence on the 4 chromosome.

No Matches-Disease primers.jpg Non disease primers.jpg