BME100 f2013:W1200 Group11 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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Contents

OUR TEAM

Name: Angelina LedesmaInitial Machine Testing
Name: Angelina Ledesma
Initial Machine Testing
Name: Shayan NaeiniResearch and Development
Name: Shayan Naeini
Research and Development
Name: Nitish PeelaProtocol Planning
Name: Nitish Peela
Protocol Planning
Name: Meera DoshiResearch and Development
Name: Meera Doshi
Research and Development
Name: Nathan DacasinInitial Machine Testing
Name: Nathan Dacasin
Initial Machine Testing

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
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The device displayed above is an OpenPCR Machine--a wooden box that has the ability to run Polymerase Chain Reaction and send the data to the computer for further analysis. A PCR machine, or Polymerase Chain Reaction machine is used to amplify target sequence of DNA by cycling through various extreme temperatures causing the DNA to split into two individual strand. Then small strands of nucleotides called primers inside each reaction mixture binds to the ends of each DNA molecule. Once cooling starts the polymerase attached to the ends of the primers and replicates the attached DNA. This allows the amplification of DNA. It can also be used to detect and/or diagnose certain diseases based on a person's DNA. The applicability of DNA fingerprinting includes helping forensic scientists when solving a murder (match DNA samples from suspects), and paternity testing.


Experimenting With the Connections
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Source: http://openwetware.org/images/7/7a/PCR_group3_.png (edited)

When we unplugged (part 3) from (part 6), the LCD screen on the machine remained lit, but did not display any information on the screen. This is because the connection between the two was unplugged and consequently no information was sent from the Circuit board to the LCD screen.

When we unplugged the white wire that connects (part 6) to (part 2), the machine was unable to take temperature readings accurately. Therefore the temperature readings were no longer displayed.

Test Run

On October 23, 2013, we tested the Open PCR machine. During this test run we had unsuccessful result since our Open PCR machine failed to operate.
Notes:

  • At the start, the LED light was working and the machine seemed to be functioning
  • During our test, the PCR machine would not display any results
  • A burning smell came from the PCR machine, and was over heating
  • No results were displayed on the computer and the LED stopped functioning

Protocols

Thermal Cycler Program
Image:Testrun212.png
1. Denature for one cycle at 95C for three minutes (Initial hold)
2. Run through 35 cycles of denaturing, annealing, and extending (the heating and cooling makes the DNA stronger)

  • Denature: Heat the DNA to 95C for 30 seconds
  • Anneal: Cool the DNA to 57C for 30 seconds
  • Extend: Heat the DNA back to 72C for 30 seconds

3. Extend the DNA at 72C for 3 minutes (to stabilize the DNA)
4. Finally, the DNA is cooled at 4C to slow down any reactions and prepares the DNA for storage

DNA Sample Set-up

Tube C+

Positive Control: Cancer DNA Template

Tube 1A

Patient 1 ID: 54764

Tube 1B

Patient 1 ID: 54764

Tube 1C

Patient 1 ID: 54764

Tube C-

Negative Control: Non-cancer DNA template

Tube 2A

Patient 2 ID: 51843

Tube 2B

Patient 2 ID: 51843

Tube 2C

Patient 3 ID: 51843

DNA Sample Set-up Procedure

  • Label each tube to reflect the controls and samples of DNA from different patients
  • Insert the DNA/primer mixes into the correct tubes and add PCR reaction mix to each tubes
  • Run OpenPCR and collect the data


PCR Reaction Mix
The pCR reaction mix is 8 tubes containing:

  • 50 µL of Taq DNA polymerase
  • 50 µL of MgCl2
  • 50 µL of dNTPs


DNA/ primer mix

  • The DNA/primer mix is 8 tubes containing 50 µL each of a different template DNA.
    • The primers (both forward and reverse) are the same for each sample

Research and Development

'PCR - The Underlying Technology'

'Components of a PCR Reaction'
Polymerase Chain Reactions require several components to ensure the effective amplification of the target DNA. For PCR to occur, template DNA is necessary. Template DNA is the sample DNA used in the PCR reaction that contains the target region, or section of DNA that can be amplified. When the template DNA is denatured, it is separated into two strands. Each strand is used as a template for the construction of a complementary DNA strand. Another component of a PCR reaction is primers. Primers are short pieces of single-stranded DNA that are complementary to a section of the template strand. In order to ensure the amplification of the target sequence, two primers are used: a forward primer and a reverse primer. A forward primer induces the elongation of the DNA strand from the 5' to the 3' direction, while the reverse primer initiates the elongation of the DNA strand from the 3' to the 5' direction. In this way, the primers begin the construction of complementary DNA strands to each strand of the template DNA at the location of the target DNA. Taq polymerase start the production of new strands of DNA by connecting to dNTP (deoxyribonucleotides triphosphate) to match with the target sequence of the DNA. Magnesium Chloride (MgCl2) is the polymerase that needs a divalent cation to function correctly. This bonds assists by being a cofactor when the polymerase and the DNA strand bind together. The polymerase with the hydroxide group is required for the bind and helps in removing the hydrogen from the deoxyribose of the nucleotides, in order to add the next nucleotide. Deoxyribonucleotides, or dNTPs, are extra bases which are building blocks for the new DNA strand.

'Steps of Thermal Cycling'
Thermal cycling initial starts at 95°C for three minutes during this period of time the enzymes are activated due to the optimal temperature that taq polymerase will become active. During this step the single stranded DNA template will also begin to disconnect. The next step is Denature which will be at 95°C for 30 second throughout this time period the hydrogen bonds with the complementary bases will begin to become distorted ultimately causing the DNA melting of the DNA template producing a single-stranded DNA molecule. Following denaturation, the DNA undergoes annealing, where primers form stable DNA-DNA hydrogen bonds with the template DNA. After the template DNA and the primers have bound, the DNA polymerase attaches to the primer-template hybrid. Annealing occurs at 57 degrees Celsius for 30 seconds. Then, extension occurs at 72 degrees Celsius for 30 seconds. During extension, the DNA polymerase enzyme synthesizes a new strand of DNA complementary to the template strand by adding dNTPs complementary to the bases of the template strand in the 5' to 3' direction. During the final step which occurs at 72 degrees Celsius for duration of three minutes any remaining single stranded DNA that is leftover will become fully extended. Also during the final step, the final hold occurs at four degree Celsius, which is when the entire reaction becomes stored.
'Base pairing'
Base pairing is the attachment of certain nucleotide bases to others to form a double-stranded DNA molecule. When primers attach to the template, base pairing allows for the creation of hydrogen bonds that allow them to stick together. The four nucleotide bases that comprise DNA are: Adenine (A), Thymine (T), Guanine (G), and Cytosine (C). Adenine and Thymine bind to each other, and Guanine and Cytosine bind to each other. This means that a "G" nucleotide base on the template strand will bind to a "C" nucleotide base on the primer. While the "A" nucleotide base on the temple strand will bind to the "T" nucleotide base on the primer. All of these nucleotides are taken by the Taq Polymerase for sequencing in the DNA template.

'Diagram of PCR'
Image:121312.jpg
There are two primers, the forward and the reverse primer, which bind to the DNA template. This image illustrates the reverse primer binding to the template. The colors of the circular components which bind to each other represent the different types of nucleotide bases in DNA: Adenine, Guanine, Cytosine, and Thymine. The Adenine base in the primer binds to the Thymine base in the DNA template, the guanine base in the primer binds to the Cytosine base in the DNA template. Likewise, the Thymine in the primer binds to the adenine in the template, and the Cytosine in the primer binds to the Guanine in the template. In the image, yellow only binds with red, and purple only binds with green, illustrating the principle of base pairing. Source : Video: "Annealing (hybridization) step of PCR." DailyMotion. Accessed 27 Oct. 2013. <http://www.dailymotion.com/video/x4mvhz_annealing-hybridization-step-of-pcr_tech>
Image:121313.jpg
This picture illustrates the binding of the forward primer to the DNA template. Just as in the binding of the reverse primer to the DNA template, the binding of the forward primer to the DNA template occurs due to the attraction of complementary nucleotide bases.Source : Video: "Annealing (hybridization) step of PCR." DailyMotion. Accessed 27 Oct. 2013. <http://www.dailymotion.com/video/x4mvhz_annealing-hybridization-step-of-pcr_tech>
Image:121314.jpg
After the forward and reverse primers have bound to separate strands of the DNA template, the DNA polymerase is ready to synthesize new DNA strands complementary to the template strands. Source : Video: "Annealing (hybridization) step of PCR." DailyMotion. Accessed 27 Oct. 2013. <http://www.dailymotion.com/video/x4mvhz_annealing-hybridization-step-of-pcr_tech>
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After the DNA polymerase has bound to the primer-template hybrid on each strand, it begins to create new strands using dNTPs as building blocks. After this occurs once, the original template strands and the newly synthesized complementary strands each separate from each other during a second cycle of denaturation. Then, forward and reverse primers anneal again, the DNA polymerase attaches once more, and complementary strands are synthesized. After multiple cycles of denaturation, annealing, and extension, the DNA polymerase is responsible for the amplification of the target region. Source : Video: "PCR." Youtube. Accessed 27 Oct. 2013. <http://www.youtube.com/watch?v=_YgXcJ4n-kQ>



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