BME100 s2016:Group2 W1030AM L4

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Contents

OUR TEAM

Bailey Gasvoda
Bailey Gasvoda
Erik Drager
Erik Drager
Ishitha Jagadish
Ishitha Jagadish
Earl Brown
Earl Brown
Destinee Martin-Karim
Destinee Martin-Karim
Logan Luke
Logan Luke

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 or samples will be cross-­contaminated
  • Cup for discarded tips
  • Micropipetter
  • OpenPCR machine: shared by two groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G2 P Positive control none
G2 N Negative control none
G2 1-1 Patient 1, replicate 1 82576
G2 1-2 Patient 1, replicate 2 82576
G2 1-3 Patient 1, replicate 3 82576
G2 2-1 Patient 2, replicate 1 67115
G2 2-2 Patient 2, replicate 2 67115
G2 2-3 Patient 2, replicate 3 67115


DNA Sample Set-up Procedure

  1. Place extracted DNA in a PCR tube.
  2. Add the first primer to the PCR tube. This primer finds one end of the DNA to be copied.
  3. Add the second primer to the PCR tube. This primer finds the other end of the DNA to be copied.
  4. Add nucleotides (A, T, G, C) to the PCR tube. These nucleotides will become the base pairs on the template DNA.
  5. Add DNA Polymerase to the PCR tube. DNA Polymerase attaches to the DNA template to add nucleotides one by one.
  6. Now the PCR tube is ready to be placed in the thermal cycler to replicate.


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

The Importance of Each Component in a PCR Reaction

  • Template DNA - The strand of DNA that contains the target sequence in which will be copied, the strand copied is the non-coding strand.
  • Primers - A short piece of single-stranded DNA that is complementary to the target sequence and serves as the starting point for DNA synthesis. They can act as forward or reverse primers and are comprised of 20 nucleotide that are attached to the end of the complementary target sequence. This allows for DNA Polymerase to attach to the DNA and start the replication process.
  • Taq Polymerase - DNA Polymerase is a complex protein attached to the target DNA sequence via primers and performs replication of the DNA strand by matching its nucleotide base pairs, creating a complementary DNA strand. During the PCR process Tay Polymerase is used due to its ability to function at high temperatures and is taken from the Thermus aquatics bacteria.
  • Deoxyribonucleotides (dNTP's) - Simple nucleotides base pairs A (adenine), T (thymine), G (guanine), and Cytosine) that are matched together with the Tay Polymerase; A-T and C-G.


Steps of Thermal Cycling

  • Initial Step to Denature Template DNA

Once the PCR tube is primed and placed in the thermal cycler, it is ready to start replicating DNA. The thermal cycler heats up to 95 degrees celsius. At this point the template DNA begins separating into two single-stranded samples of DNA. The DNA is completely separated after 3 and half minutes.

  • Anneal and Attach Primers

At this point, the thermal cycler lowers the temperature to 57 degrees celsius. This allows DNA primers to attach to the template DNA. The primers are segments of nucleotides designed to pair with one end of the DNA to be studied.

  • Extend the DNA Strand and Repeat

The thermal cycler heats up to 72 degrees celsius to allow DNA Polymerase to attach to the single stranded DNA with primers. The DNA Polymerase begins adding complementary nucleotides one by one to the template DNA following the primer. This continues until the DNA strand is fully replicated. The cycle can now be repeated to increase the amount of target DNA to any desired amount. After the desired amount is achieved, it is stored at 4 degrees celsius.


Original Picture of PCR Steps

NOTE: For some reason, this picture keeps formatting itself horizontally instead of vertically. Sorry for the inconvenience!



Components and Functions

Template DNA - The strand of DNA that contains the target sequence in which will be copied, the strand copied is the non-coding strand.

Primers - A short piece of single-stranded DNA that is complementary to the target sequence and serves as the starting point for DNA synthesis. They can act as forward or reverse primers and are comprised of 20 nucleotide that are attached to the end of the complementary target sequence. This allows for DNA Polymerase to attach to the DNA and start the replication process.

Taq Polymerase - DNA Polymerase is a complex protein attached to the target DNA sequence via primers and performs replication of the DNA strand by matching its nucleotide base pairs, creating a complementary DNA strand. During the PCR process Tay Polymerase is used due to its ability to function at high temperatures and is taken from the Thermus aquatics bacteria.

Deoxyribonucleotides (dNTP's) - Simple nucleotides base pairs A (adenine), T (thymine), G (guanine), and Cytosine) that are matched together with the Tay Polymerase; A-T and C-G.

Steps of Thermal Cycling


Initial Step to Denature Template DNA Once the PCR tube is primed and placed in the thermal cycler, it is ready to start replicating DNA. The thermal cycler heats up to 95 degrees celsius. At this point the template DNA begins separating into two single-stranded samples of DNA. The DNA is completely separated after 3 and half minutes.

Anneal and Attach Primers At this point, the thermal cycler lowers the temperature to 57 degrees celsius. This allows DNA primers to attach to the template DNA. The primers are segments of nucleotides designed to pair with one end of the DNA to be studied.

Extend the DNA Strand and Repeat The thermal cycler heats up to 72 degrees celsius to allow DNA Polymerase to attach to the single stranded DNA with primers. The DNA Polymerase begins adding complementary nucleotides one by one to the template DNA following the primer. This continues until the DNA strand is fully replicated. The cycle can now be repeated to increase the amount of target DNA to any desired amount. After the desired amount is achieved, it is stored at 4 degrees celsius.


Condensed Version of Components and Functions




SNP Information & Primer Design

Background: About the Disease SNP

  • Nucleotide: Nucleotides form the basic structural unit of nucleic acids such as DNA and consist of a nucleotide linked to a phosphate group.

There are four nucleotides used in DNA, A (adenine), T (thymine), G (guanine), and Cytosine) where A matches with T and G matched with C.

  • Polymorphism: In biology and Zoology polymorphism is used to clarify that two different forms of species arose from the same genotype.

This explains the definition of polymorphism for clear as it is the presence of genetic variation or different within a population of the same species, in which natural selection can occur.

The single nucleotide polymorphism tested (rs36686) was found to be in Homo Sapiens variation. It os found on the B3GNT3 gene on chromosome 19:17811986 and has no clinical significance. Although, this SNP is linked to non-Hodgkin Lymphoma which is a cancer in the lymphocytes cells located in the immune system.


Primer Design and Testing B3GNT3 enzyme stands for beta-1,3-N-acetylglucosaminyltransferase and is a type II transmembrane protein with a single un-cleaved anchor. B3GNT3 is involved in the biosynthesis of poly-N-acetyllactosamine chains and the backbone structure of dimeric sialyl Lewis a. Its major role though is L-selectin ligand biosynthesis, lymphocyte homing and lymphocyte trafficking. The first unique terms in the gene ontology are N-acetyllactosaminide beta-1,3-N-acetylglucosaminyltransferase activity; beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,3-N-acetylglucosaminyltransferase activity; galactosyltransferase activity.

An allele is one of two or more alternative forms of a gene formed by a mutation found on the same chromosome. The disease associated allele contains CAC sequence and is at the numerical position of the SNP at 17811986. The non-disease forward primer (20 nt) for this SNP in 5’ -GTGCGGGCTCCATCGCAACG- and the numerical position exactly 200 bases to the right id the disease SNP is 17812186. The non-disease reverse primer (20 nt) is 5’ -GGAGGAAGGTGTCGCCCCTT and after the final base of the disease forward primer was changed so that it is identical to the disease SNP base it was 5’ -GTGCGGGCTCCATCGCAACT. The final disease forward primer (20 nt) is 5’ -GTGCGGGCTCCATCGCAACT and the final disease reverse primer (20 nt) is 5’ -GGAGGAAGGTGTCGCCCCTT. These primers were tested in a PCR generator and the results are represented below.


Non-disease Primers


Disease Primers




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