BME100 f2013:W1200 Group11 L4

BME 100 Fall 2013	Home People Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3 Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6 Course Logistics For Instructors Photos Wiki Editing Help
OUR TEAM Name: Angelina Ledesma Initial Machine Testing Name: Shayan Naeini Research and Development Name: Nitish Peela Protocol Planning Name: Meera Doshi Research and Development Name: Nathan Dacasin Initial Machine Testing LAB 1 WRITE-UP Initial Machine Testing The Original Design A PCR machine, or Polymerase Chain Reaction machine, produces copies of DNA which are then used to produce thousands, even millions, of copies of strands from a certain segment of DNA using a heating cycle. It can also be used to detect and/or diagnose certain diseases based on a person's DNA. It can also be used as a DNA fingerprinting machine, which is critical to forensic scientists when solving a murder (match DNA samples from suspects), and paternity testing. Experimenting With the Connections 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. When we unplugged the white wire that connects (part 6) to (part 2), the machine Test Run We first tested the Open PCR machine on October 23, 2013. During this test run we had unsuccessful result, as our Open PCR machine failed to operate. Protocols Thermal Cycler Program 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 Step 1 Step 2 Step 3... PCR Reaction Mix What is in the PCR reaction mix? DNA/ primer mix What is in the DNA/ primer mix? 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 take by the taq polymerase in for sequencing in the DNA template. 'Diagram of PCR' 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> 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> 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> 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>