BME100 f2015:Group16 1030amL4

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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

Steven Rojo
Jacob Baca
Sudarshan Kandel
Aditya Konduri
Nicolas March
Javon Boaz

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat
  • Disposable gloves
  • PCR reaction mix,
  • 8 tubes
  • PCR reaction mix containing 50 μL of the following: Taq DNA polymerase, MgCl2, and dNTP’s
  • 8 tubes,
  • DNA/ primer mix containing 50 μL of a different template DNA but the same forward primer and reverse primer
  • A strip of empty PCR tubes
  • Disposable pipette tips
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine ( which is shared by two groups)


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G# + Positive control none
G# - Negative control none
G# 1-1 Patient 1, replicate 1 17821
G# 1-2 Patient 1, replicate 2 17821
G# 1-3 Patient 1, replicate 3 17821
G# 2-1 Patient 2, replicate 1 75677
G# 2-2 Patient 2, replicate 2 75677
G# 2-3 Patient 2, replicate 3 75677


DNA Sample Set-up Procedure

1) In order to perform a Polymerase Chain Reaction (PCR), DNA must be collected from cells from a source such as hair follicles.

2) Following this, the DNA should be extracted from the source into a designated PCR tube

3) Then, the initial primer should be extracted and inserted into the same designated PCR tube

4) The second primer is then extracted and released into the same designated PCR tube

5) Nucleotides are then added to the PCR tube in order to provide the needed building blocks when the DNA replicates

6) The final thing to be added is DNA polymerase that commences the matches the nucleotides in order to make the DNA copies

7)After everything has been released into the test tube, place the PCR tube into the 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, andExtend at 72°C for 30 seconds

FINAL STEP: 72°C for 2 minutes

FINAL HOLD: 4°C





Research and Development

PCR - The Underlying Technology


What is the function of each component of a PCR reaction?

  • Template DNA​: Predetermined form that synthesis DNA polymerase and artificial DNA into a single molecule.
  • Primers​: Short pieces of DNA that are made in a laboratory. Being custom made, they can have any sequence of nucleotide you would like.
  • Taq Polymerase​​: An enzyme that assembles nucleotides into new strands of DNA.
  • Deoxyribonucleotides (dNTP’s)​: They create copies of the desired segments of DNA that are targeted.

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

  • INITIAL STEP (95°C for 3 minutes): The DNA double helix separates into two separate single-stranded DNA molecule.
  • Denature​at (95°C for 30 seconds):The DNA polymerase in our bodies breaks down.
  • Anneal​at (57°C for 30 seconds): The single strands start to join together with primer sequences.
  • Extend​at (72°C for 30 seconds): DNA polymerase is activated.
  • FINAL STEP (72°C for 3 minutes): Complementary nucleotides are joined to the primer on the DNA.
  • FINAL HOLD (4°C): Storage of the DNA is initiated for a definite amount of time.

Which base anneals to each base? A to T T to A G to C C to G

During which two steps of thermal cycling does base-pairing occur? Explain your answers. This process occurs during Annealing and Extension, where the base pairs are polymerized and joins with the primer sequences. At these two points of the process, the amount of DNA is doubled and is prepared for the entire process to repeat itself.

SNP Information & Primer Design

Background: About the Disease SNP SNP (Single Nucleotide Polymorphism) is the occurance of a nucleotide (adenine, thymine, guanine, or cytosine) change to another nucleotide. An example of this would be if a guanine changing into a thymine. The result could be minimal or could result in a horrific disease, like Alzheimer's or Crohn's disease, to occur in the patient.


Primer Design and Testing

What is a nucleotide? The structural components, or building blocks, of DNA and RNA. It consists of a base (adenine, thymine, guanine, and cytosine) plus a molecule of sugar and one of phosphoric acid.

What is a polymorphism? The variation of a certain gene in a specified population

What species is this variation found in? Homo Sapiens

What chromosome is the variation located on? 16:89919736

What is listed as the Clinical significance of this SNP? Pathogenic

Which gene(s) is this SNP associated with? MC1R

What disease is linked to this SNP? Melanoma, Parkinson's, Crohn's

What does MC1R ​stand for? Melanocortin 1 Receptor

What is the function of MC1R? Melanocortin receptor activity, Hormone Binding, G-Protein coupled peptide receptor activity

Write the first three unique terms you see.

What is an allele? A variant form of a gene.

The disease-associated allele contains what sequence? CGG->TGG

The numerical position of the SNP​ is: 89919736

Non-disease forward primer: 5'-CAGCATCGTGACCCTGCCGC

The numerical position exactly 200 bases to the right of the disease SNP​is: 89919936

Non-disease reverse primer: 5'-CTTGTGGAGCCGGGCGATGC

Disease forward primer: 5'-CAGCATCGTGACCCTGCCGT

Disease reverse primer: 5'-CTTGTGGAGCCGGGCGATGC

Why is there no match for the Disease-specific primer we created? There were no matches for the created primer because the specific portion of the DNA that we attempted to replicate could actually result in a replication. That it has no matches also establishes that no other extraneous portions of the DNA are replicated resulting in a originality between the primer and the DNA strand. This is assured by how it will only be replicated to the targeted SNP rather than the extraneous DNA.

Results for a) >chr16:89986125+89986344 220bp CAGCATCGTGACCCTGCCGC CTTGTGGAGCCGGGCGATGC CAGCATCGTGACCCTGCCGCgggcgcggcgagccgttgcggccatctggg tggccagtgtcgtcttcagcacgctcttcatcgcctactacgaccacgtg gccgtcctgctgtgcctcgtggtcttcttcctggctatgctggtgctcat ggccgtgctgtacgtccacatgctggcccgggcctgccagcacgcccagg GCATCGCCCGGCTCCACAAG

Results for b) No matches to cagcatcgtgaccctgccgt cttgtggagccgggcgatgc in Human Feb. 2009 (GRCh37/hg19)