New School DNA sequencing (with lasers!): Bacterial ID Lab

Bacterial ID Lab

This lab will help you learn the basics of modern DNA sequencing techniques and how they are applied in diagnosing infectious diseases. Completing this virtual lab and answering the following questions will allow you to post additional evidence for Portfolio Standard 2: Plan and Carry Out Scientific Investigations.

First, go to:

http://www.hhmi.org/biointeractive/vlabs/bacterial_id/index.html or the iPad app

In this virtual lab you will assume the role of a lab technician in a modern molecular biology laboratory.  As such, you are responsible for providing lab results to medical doctors for use in diagnosing their patients.  Be sure to follow the steps of the procedure in order and to make use of the notes on the right side of the computer screen.  As you work through the lab, answer the following questions:

1. As the medical technician in charge of this investigation, what are you trying to determine about the tissue sample provided to you?

i am trying to determine a bacteria sample

2. How did you prepare the DNA to be used in this investigation?

first i put on the gloves then went to the culture sample and got the wire loop and extracted a colony form the culture sample. then i transferred the sample to the microcentrifuge tube. then i added digestive enzymes. then i let it sit for a couple of hours. then i put it in a heat bath to activate the digestive enzymes. then i put it into a counter balance to remove any cellular debris. then i transferred to mixture into the pcr tubes. and that's how i prepared this dna for this investigation.

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.

4. Summarize the technique used to purify the PCR product.

The tube should now contain many copies of 16s rDNA, each about 1,500 base pairs (bp) long. At this time, it is prudent to run a gel to confirm that the PCR reaction worked. The gel should contain three lanes: one for the negative control (i.e., water), which should not have a product unless the water was contaminated; another for positive control (PCR product of a known DNA sequence) to make sure that the PCR itself worked; and the last lane for your sample.

If you are confident that the PCR worked, you can proceed to purifying the PCR product. Running a gel is actually one method of purification. Once the PCR product is in the gel, you can cut out the band corresponding to the PCR product and isolate the DNA from the gel. Nowadays, you can buy compact microfilters to filter the DNA from the PCR tube without running a gel. We will use such microconcentrator columns in our procedure:

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.

The final collection tube should now have many pieces of 1,500bp-long 16S rDNA, with a very small amount of longer DNA strands (which are contaminants).

5. What is produced during the sequencing prep PCR run? Use the animation and notebook as needed in thinking through your answer.

6. Describe how the automatic sequencer determines the sequences of the PCR products.

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)?