BME100 f2013:W900 Group17 L4

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
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Contents

OUR TEAM

Name: Abrar BakhshRole: Protocol Planning
Name: Abrar Bakhsh
Role: Protocol Planning
Name: Jeremy BeckerRole: Open PCR Machine Testing
Name: Jeremy Becker
Role: Open PCR Machine Testing
Name: Luis HernandezRole: Research and Development
Name: Luis Hernandez
Role: Research and Development
Name: Alison LlaveRole: Open PCR Machine Testing
Name: Alison Llave
Role: Open PCR Machine Testing
Name: Naaz MaududiRole: Research and Development
Name: Naaz Maududi
Role: Research and Development

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design of the PCR machine
Image:OpenPCR17.jpg

The device displayed in this image is an Open PCR machine which is a machine that conducts a polymerase chain reaction which in turn is a reaction that amplifies a given piece of DNA, or in other words makes many more copies of it. This is done inside the machine through a multi-step heating and cooling process the first of which is to heat the DNA in order to have it split apart from the two helix form into individual chains. Here primers, small strands of nucleotides, inside the reaction mixture attach to the ends of the individual DNA chain. Then when cooling begins to happen the polymerase, an enzyme, in the reaction mixture attaches to the ends of the primers and begins replicating the individual chains of DNA that it's attached to using floating nucleotides in the reaction mixture. Finally, the new DNA chains made as copies fuse together as do all the DNA chains present. This represents just one cycle of many taken to produce a multitude of copies of the DNA that is being amplified. This whole process takes place inside an area called the thermal block that holds tubes with the reaction mixtures which are heated and cooled according to the process. A lid on top of this area prevents condensation of the water of the reaction mixture. Another feature of this machine is its ability to interface with a computer via a USB cable which allows the monitoring of the machine's progress through cycles like the aforementioned one on the computer. However, if one wishes to view this information directly on the machine this is also possible due to the attached LCD screen.


Experimenting With the Connections on the machine

Image:OpenPCRModel.jpg

When part three, or the screen, was unplugged from part 6, the control circuit, the screen ceased to function and would not turn on until the connection was restored because without the conncection the screen was not receiving a signal from the control circuit. It is important for the screen to continue to function since it displays the number of cycles performed by the achine and the temperature.

When the white wire connecting part 6, the control circuit, to part 2, the thermometer, was disconnected the temperature measurement changed suddenly from 23.6 degrees Celsius to -40 degrees Celsius. Essentially, the temperature readout began to be extremely inaccurate.

The Test Run

The PCR machine was first used to run through a test cycle on October 23, 2013 at 9:56 am and stopped at 11:16 am for a total run of one hour and twenty minutes. During this time the machine ran without any major problems and in fact was very quiet throughout the cycles it went through. Also, the time to completion displayed on the computer monitor through the program was always within reasonable parameters given the average time it takes for the PCR machine to complete its ideal number of cycles. Given the time that the PCR machine in this experiment was allowed to operate, one hour and twenty minutes, it was able to complete 25 cycles in total with thirty six minutes being left of operation time as inficated by the timer.




Protocols

Thermal Cycler Program
35 cycles of the following occurs with a duration 30 seconds each.

  1. Stage 1: The Initial Step of the program is set to 95 degrees C for 3 minutes, or 180 seconds. At this stage, the template DNA begins to denature. 35 cycles of the following occurs with a duration 30 seconds each.
  2. Stage 2:First, the temperature starts out at 95 degrees C, keeping constant from the previous stage, during which double strands of DNA begin to separate. This lasts for thirty seconds.
  3. Stage 3:Then during the next 30 seconds, the temperature decreases to 57 degrees C, which allows primers to attach to the ends of the target DNA sequence.
  4. Stage 4:Finally for the next 30 seconds, the temperature increases up to 72 degrees so that the DNA polymerase is activated and begins replicating the DNA by building new chains from dNTPs in the reaction mixture.
  5. Stage 5:During this stage the temperature is held at 72 degrees Celsius in order to ensure that any remaining single stranded DNA molecules are fully extended by the DNA polymerase.
  6. Stage 6:The Final Hold is set to 4 degrees C for 3 minutes to stop the polymerase chain reaction and allow for short term preservation of reaction.

DNA Sample Set-up

Positive Control: Cancer DNA Template, label:M1Patient 1, ID:80926, label:M2Patient 1, ID:80926, label:M3Patient 1, ID:80926, label:M4


Negative Control: Non-Cancer DNA Template, label:F1Patient 2, ID:83588, label:F2Patient 2, ID:83588, label:F3 Patient 2, ID:83588, label:F4

DNA Sample Set-up Procedure

  1. Step 1: 8 tubes containing 50 microliters of PCR reaction mix were given
  2. Step 2: Reaction tubes were labeled according to the table above
  3. Step 3: Using a micropipette, 50 microliters of each DNA sample mixture were added into its respective reaction tube
  4. Step 4: The reaction tubes were laced into the thermocycler, and the thermocycler was activated to run according to the program details stated above.


PCR Reaction Mix

  • In the PCR reaction mix is 50 μL each of the following: taq DNA polymerase, Magnesium Chloride (MgCl2), and dNTPs(deoxyribonucleotides). Taq DNA polymerase is of course the enzyme responsible for the synthesizing of new replicated DNA molecules usig base pairing with respect to the template strand parallel to the polymerase. The DNA polymerase is able to form new DNA molecules by using the dNTP building blocks spread throughout the reaction mixture. Finally, Magnesium chloride is also an important component as it is a required cofactor for the taq DNA polymerase.


DNA/ primer mix

  • In the DNA/ primer mixture is 50 μL each of a template DNA, which varies for each sample, forward primers, and reverse primers. The template DNA is the DNA molecule containing the sequence of DNA that is to be amplified while the forward primers are the short DNA sequences complimentary to the starting sequence of the sequence or piece of DNA being amplified and the reverse primers are the opposite in a way as they are short DNA sequences complimentary to the ending sequence of the piece of DNA being amplified.





Research and Development

Template DNA, Primers, and dNTPs Since polymerase chain reaction (PCR) is a process designed to amplify DNA, a certain strand of DNA containing a specific sequence of nucleotides must be the target of this amplification. This strand is called the template DNA and it is this DNA that undergoes the PCR process of replication. In general, this process works similarly to the natural cellular way of replicating DNA in that the DNA molecule is first split into its component chains from the usual double helix structure and then copied using polymerase, an enzyme that copies DNA as it moves doen a chain after attaching to a primer. However, the difference is that instead of an enzyme splitting the DNA double helix the double helix experiences being denatured due to the amount of heat produced in PCR's first step. This denaturing means that the helix splits into two chains. Shortly after, however, cooling begins to occur and primers, which are short strands of nucleotides, begin to attach to their complementary sequences on the DNA chains. This attachment of primers is necessary for DNA replication as polymerases can only add nucleotides to existing strands of DNA, something which a primer is. Being on the subject of DNA and especially replication, it should be made clear that replication of DNA and formation of new identical DNA strands is only possible because of the deoxyribonucleotides (dNTPs) present throughout the reaction mixture. These dNTPs are what the DNA polymerase uses as building material for the construction of replicated chains.

Taq Polymerase, cofactor, and base pairing Going back to the PCR process which began to be described in the previous paragraph, it is found that after cooling begins primers attach to the individual strands of DNA. However, what is not yet mentioned is that once it cools a sufficient amount, the enzyme taq polymerase is activated and begins to attach to the ends of primers. This enzyme is what is responsible for synthesizing new strands of DNA by adding dNTPs from the reaction mixture to the ends of primers according to the base pairing rules with regard to the template strand the polymerase is working parallel to. Before mentioning the exact base pairing rules, one more component of PCR deserves to be mentioned which is Magnesium Chloride, a cofactor to the taq polymerase. This magnesium chloride should be kept at optimum concentration for every template in order to work effectively as a cofactor. Finally, it is worth mentioning the base pairing rules, especially since they are a big part of the actual work of replication performed by the polymerase. How base pairing works amongst the four dNTPs is that Adenine pairs with thymine with the reverse also being true and that Cytosine pairs with Guanine with the reverse also being true.

PCR process Initially, after the PCR machine is ready and the cycle begins the first step consists of the thermal block heating to 95 degrees Celsius as is required when using hot start PCR and of course a heat activated polymerase, or rather a polymerase which becomes inactive at a certain range of temperatures and then once again becomes active at a different range. The second step then consists of the temperature staying at 95 degrees Celsius for three minutes so that the DNA in the reaction mixture denatures. After, there is a cooling down to 57 degrees for thirty seconds during which time primers begin to attach or anneal to the separated DNA strands. Next there is another heating and raise of temperature for thrity seconds but only to 72 degrees. It is at this point that polymerases attach to the ends of primers and begin replicating DNA strands thorough the following of base pairing rules and the usage of dNTPs spread through the reaction mixture. Finally, a last step which specifies keeping the temperature at 72 degrees Celsius is performed in order allow the extension of any remaining single stranded DNA molecules by the taq polymerase. Though this is the last step one more measure can be taken in order to allow for the storage of this reaction for a limited time. This would be to drop the temperature to four degrees Celsius. It is this step that is put under the appropriate name final hold.


This image represents the process of PCR and how cancerous DNA will undergo an exponential growth I11-50-PCR.jpg pierce_18_19_large_2.jpg

https://www.google.com/searchq=process+of+pcr&source=lnms&tbm=isch&sa=X&ei=yFRwUqGTOIKxiwKY64CADw&sqi=2&ved=0CAcQ_AUoAQ&biw=1951&bih=961#q=process+of+pcr+amplification&tbm=isch&facrc=_&imgdii=_&imgrc=DFzRQQKNLcbzVM%3A%3BpKI0UaXZHG8oRM%3Bhttp%253A%252F%252Fwww.nature.com%252Fscitable%252Fnated%252Fcontent%252F19492%252Fpierce_18_19_large_2.jpg%3Bhttp%253A%252F%252Fwww.nature.com%252Fscitable%252Ftopicpage%252Fthe-biotechnology-revolution-pcr-and-the-use-553%3B700%3B323




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