This lab we did virtually at this website. Basically it took us through a lab as if we were the

1. As the medical technician in charge of this investigation, what are you trying to determine about the tissue sample provided to you? You are trying to determine wether or not the patient contains a disease or not.

2. How did you prepare the DNA to be used in this investigation?  First we had to grow a colony of DNA to test it with. Since we use other enzymes during the experiment, we we have to dispose of the proteolytic enzymes before moving on. The enzymes are removed by heating the sample in a water bath at 100°C. In doing so, the cellular debris is sent down in the centrifuge and appears as a solid deposit (a small pellet) at the bottom of the tube. The DNA is contained in the supernatant (the liquid), which is then transferred to the PCR tube.

3. Describe how PCR is used to make copies of DNA sequences. Use the animation and notebook entries in the PCR Amplification step to guide your answer. Note that you may replay the animation as needed. The first step is to melt the sample. This separates the two DNA chains in the double helix by heating the vial containing the PCR reaction mixture to 95°C for 30 seconds. The primers are incapable of binding to the DNA strands at such a high temperature, so the vial is cooled to 60°C. At this point, the primers bind to the single-stranded DNA. The reason the two separated strands of DNA do not reanneal is that there is a large excess of primers in the solution; therefore, it’s more likely for the DNA strands to bind to the primers instead of to each other. The final step is extending the sample again. This step is to allow the DNA polymerase to extend the copy DNA strand by raising the temperature to 70°C for 45 seconds.

4. Summarize the technique used to purify the PCR product. The purification process can only be done had you correctly done the phase to prior to the current one. During this step you will use compact micro filters to filter the DNA. In specific we used micro concentrator columns to do so. The procedure to this phase are listed as so; 
   1. Insert the microconcentrator column of appropriate size into a collection tube.
   2. Add 400 µL of buffer to the column.
   3. Add the entire PCR content (~100 µL) to the column.
   4. Spin the column at 3,000 rpm in a fixed-angle centrifuge for 15 minutes.
   5. The PCR product should be trapped in the column while the collection tube should contain all the primers, nucleotides, and other small compounds that we no longer need. Remove the collection tube and discard it.
   6. Invert the column and attach it to a new collection tube.
   7. Add 50 µL of buffer to the inverted column. This step should loosen the DNA from the column into the collection tube.
   8. Spin the inverted column at 3,000 rpm for 2 minutes to collect the sample in the collection tube. Discard the column.

5. What is produced during the sequencing prep PCR run? Use the animation and notebook as needed in thinking through your answer. In the prep PCR run, the varied amount of tubes (in this case 12, six blue and six green), are ran through the PCR machine to attempt to produce strands of DNA the same as the starting sample. In the second run the goal is to produce many strands of DNA with similar sequences.

6. Describe how the automatic sequencer determines the sequences of the PCR products. An automatic sequencer performs gel electrophoresis on the DNA in each tube. Gel electrophoresis is a method to separate molecules based on differences in size.

7. What does BLAST stand for? “Basic Local Alignment Search Tool”

8. What conclusions did you make using the results of the BLAST search?  Did these conclusions support a clinical diagnosis for the patient (what disease did they have)? The disease conclusions that I got matched up to be a 98% match to a disease. After I “BLAST”ed the DNA sequence the disease that it matched to was called Yersinia Enterocolitica. 

In this blog I’m going to cover a thing called central dogma. The view of central dogma of biology states that the genetic information that is hard wired into strands of DNA is transcribed into individual transportable cassettes. These cassettes are composed of messenger RNA (mRNA).Each cassette of mRNA contains a specific code for synthesis that will make a specific protein. Click here to view a video going over central dogma that uses a bunch of little spaceships and really cool music. Below I’m Going to insert a file uploaded by my teacher Introduction_to_Central_Dogma that gives a brief three page review of the definition of central dogma I gave above.

These are some terms that relate to central dogma:

DNA- Deoxyribonucleic acid

RNA- Ribonucleic acid

mRNA- Messenger ribonucleic acid

Protein- Compounds or chains of amino acids

Transcription- Synthesis of RNA under the direction of the DNA.

Translation- mRNA is decoded by a ribosome to produce a specific amino acid chain.

Nucleotides- A compound of nucleoside that goes with a certain phosphate group.

Amino Acids- The basic constituents of proteins.