BME100 s2015:Group2 12pmL4

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OUR TEAM

Name: Levi Riley
Name: Alexandria Clark
Name: Blossom Mendonca
Name: Alina Kilic
Name: Trevor Douglass
Name: Andre Dang

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/gotaqcolorless-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 re-use disposable pipette tips or

samples will be cross-contaminated

  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine: shared by two groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G2 + Positive control none
G2 - Negative control none
G2 1-1 Patient 1, replicate 1 58575
G2 1-2 Patient 1, replicate 2 58575
G2 1-3 Patient 1, replicate 3 58575
G2 2-1 Patient 2, replicate 1 86157
G2 2-2 Patient 2, replicate 2 86157
G2 2-3 Patient 2, replicate 3 86157


DNA Sample Set-up Procedure

  1. The group obtained all materials listed in the protocol section
  2. The group took the empty strip of PCR tubes given & cut them in half so that there were two strips of four linked tubes. This was done so that the PCR tubes would fit into the PCR machine because the machine only fits up to four in a row.
  3. The group labeled the empty PCR tubes using the labels from the table listed above. It was labeled using a black marker which was also erasable so that it could be removed afterwards.
  4. The tubes were transferred to a rack to secure them while the micro-pipette was used.
  5. In each of the eight tubes, 50 micro liters of PCR reaction mix was transferred into each tube. Then, in the tube labeled for positive control, 50 micro liters of solution was transferred. Similarly, 50 micro liters of solution was pipetted into the test tube labeled for the negative control. Between each use of the pipette, the tip was changed out to avoid contamination.
  6. After this was completed, 50 micro liters of the sample from patient 1 was transferred into it's appropriately labeled test tube. This same process was repeated for the remaining samples of patient one and patient two. The group took note to change the tip after each use of the pipette to avoid contamination.
  7. The group took note that all the labeled tubes to be transferred into the PCR machine contained 100 micro liters of material.
  8. The group closed all the lids for the PCR Reaction tubes (eight total).
  9. The group then asked the TA for help and placed the labeled PCR tubes into the appropriate rows for the PCR machine. A second group placed their PCR tubes into the same machine, and then the machine was set to the specifications listed below in the OpenPCR Machine section below.


OpenPCR program

  • HEATED LID: 100°C
  • INITIAL STEP: 95°C for 2 minutes
  • NUMBER OF CYCLES: 35

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

Components and Their Functions in a PCR Reaction

For the PCR Reaction, there are four main components. These components are: Template DNA, Primers, Taq Polymerase, Deoxyribonucleotides (dNTP’s). The template DNA is the target DNA which the PCR process is amplifying. This DNA has a specific sequence that is to be amplified by the PCR process. But before it can be amplified (replicated over and over again), the template strand must first be separated. This is done by adding heat to the system which separates the double-stranded template DNA into individual strands. After they are separated, a primer is used to locate the specific sequence of DNA that is to be amplified. Primers themselves are short segments of DNA that bind to a denatured (separated) strand and signal where the DNA is to start being replicated. Primers have to be selected carefully to ensure that they bind to the site of interest and signal to replicate the section of DNA that follows. After the primer has bound to its respective place on the denatured DNA, Taq Polymerase – an enzyme – comes in and reads the DNA sequence following the primer. The Taq Polymerase matches the template DNA with its respective base pair. In DNA, there are four bases – Adenosine (A), Thymine (T), Cytosine (C), and Guanine (G) – and each bas has its respective partner. A’s bond to T’s and C’s bond to G’s. That being said, the Taq Polymerase matches the Template DNA with its appropriate base pair. These base pairs are free floating in solution in the form of Deoxyribonucleotides. Deoxyribonucleotides are the individual bases – A, T, C, G – and the Taq Polymerase grabs the respective base and pairs it with its proper pair in order to create a two double stranded DNA molecules based upon the original template strand that was placed in solution. This process is repeated over and over again in PCR to create many copies based upon the target sequence of the template DNA.


Components and How They Play A Role In Thermal Cycling

At the initial step of the PCR reaction, the machine with the samples is brought to 95 degrees Celsius for three minutes. This helps the bonds in the double stranded template DNA start to loosen as the heat is applied. When an additional 30 seconds at 95 degrees Celsius is added, the template DAN completely separates – or Denatures – and creates two single-stranded DNA molecules. DNA does not like to stay split up for long which is why such a high heat is necessary for separating the DNA and why the next step has to follow quickly. In the Annealing step of the PCR process, the primers quickly bind to their respective sites on the two single stranded DNA molecules and this no longer allows the DNA to join back together and form a double helix. Also, this step is carried out for 30 seconds at 57 degrees Celsius. It must have a lower temperature because when the system is at 95 degrees Celsius, nothing can bind due to the high temperatures. The 57 degrees Celsius allows the primer to come in and bind to its appropriate site. In the Extending section, the Taq Polymerase is activated by the change in heat and it locates the newly bound primer and then reads down the DNA and adds the respective Deoxyribonucleotide base pairs to create two double stranded DNA molecules from the original one double stranded DNA molecule. This process is carried out at 72 degrees Celsius for 30 seconds. The Taq Polymerase acts quickly in reading the DNA and adding the appropriate base pairs. When the Taq Polymerase reaches the end of the DNA strand, it falls off (detaches from) the end of the DNA strand and the replication is complete. The Final Step of the PCR cycle is then carried out which hold the temperature at 72 degrees Celsius for 2 minutes and ensures that the bonds are properly formed in the DNA double helix. This concludes one cycle of the PCR machine; however, the same process (initial step through final step) is repeated 34 more times to make a total of 30 PCR reactions which take place during the amplication of DNA via PCR. After the 35 cycles have been carried out, there is one final step that needs to be observed. The system is brought to a final hold at 4 degrees Celsius for a short period of time. This is done to ensure that the DNA will not split apart again by bringing to such a cool temperature where it is not physically possible for the DNA to split apart.


Base Pairing

As described earlier, DNA is composed of four pairs of bases: Adenine (A), Thymine (T), Cytosine (C), and Guanine (G). Each Base pairs up with only one other kind of base. This makes it so that the Genetic code is carried out by making sure one base matches with another. For the base pairs, Adenine binds with Thymine and Cytosine binds with Guanine.


Base Pairing In The PCR Cycle

In the PCR cycle, base pairing occurs in two places. It first occurs in the Annealing step where primers bind to their respective section on the denatured, single-stranded DNA. This occurs because, as described earlier, primers are short fragments of DNA meaning that since they are a specific sequence, they can only match up with the DNA that holds the base pairs which match up. Base pairing also occurs in the Extending step where Taq Polymerase brings in the Deoxyribonucleotides freely floating in solution and binds them to their respective base pair based upon the original DNA strand.