INTRODUCTION TO MOLECULAR

BIOLOGY AND GENETICS

MBGE102

Respected Asst. Prof. Dr. Ayşe Seyer

Zahra Arang 21814225

 BACTERIAL IDENTIFICATION LAB HANDOUT

INTRODUCTION

1.What is the overall purpose of this virtual lab?

The purpose of this virtual lab is to familiarize me with the science and techniques used to
identify

2.What are the four basic steps involved in this bacterial identification lab?

Prepare a sample from a patient and isolate whole bacterial DNA.

Make many copies of the desired piece of DNA.
Sequence the DNA.
Analyze the sequence and identify the bacteria.

3.What is "16S rDNA," and how is it used to identify species of bacteria?

16S r DNA is the DNA used for identifying bacteria. It is the region that codes for a small subunit
of theribosomal RNA or 16S rRNA. Using it to identify bacteria species requires that the sample
go through adatabase of all the known 16S rDNA sequences until a matching sequence can be
found.
PART 1: SAMPLE PREPARATION

4.As the pathology lab technician, what is your task in this virtual lab?
As the pathology lab technician, my task in this lab is to identify a bacterial sample provided by a
clinician

5. Extracting DNA involves which initial step?

The initial step to extracting DNA is to pick up a single colony of bacteria and drop it into a
microcentrifuge tube.

6. What is the wire ring used for?

The wire ring is used to pick up a single colony of the grown bacterial colonies and transfer it to
the microcentrifuge tube.

7. Why are the proteolytic enzymes necessary?

Extracting DNA from bacteria involves dissolving the cell wall using a digestive buffer. This buffer
contains proteolytic enzymes which eat away at the cell wall over an extended period of time.

8. Why do you then need to inactivate the proteolytic enzymes and how do you do it?
Following the use of a digestive buffer and thus proteolytic enzymes, we will be using other
enzymes which is why we have to inactivate the proteolytic enzymes. To do so, the sample is
placed in a heated water bath at 100 degrees Celsius.

9. After removing the enzymes, why do you spin the sample in the centrifuge?
With the enzymes removed, the next step is handling the cellular debris which is why the sample
is spun in the centrifuge.

10. a. What is the pellet?

The pellet is the solid pellet at the bottom of the sample tube that use to be the cellular debris.

b. What is the supernatant?

The supernatant is the liquid.

c. Where is the DNA?

The DNA from the sample is contained in the supernatant
PART 2: PCR AMPLIFICATION
Go on to Part 2 and work through the PCR steps. Be sure to read the information in the notebook,
including “What is PCR?”

11. What does “PCR” stand for and what is the purpose of PCR?
PCR stands for Polymerase Chain Reaction and is a technique used to allow many copies of DNA
to be made.

12. Summarize the process of PCR in a diagram. Include all the steps, labeled and in the right order. (If
you are completing this handout online, draw the diagram on a piece of paper, take a photo, save the
image as a PDF, and upload it in the space below.)

Add the Master Mix and answer the following questions:

13. What does the Master Mix contain?

The Master Mix contains water, a buffer to keep the mixture at the correct pH for the PCR
reaction, large quantities of nucleotides adenine, cytosine, guanine and thymine, large quantities
of oligonucleotide DNA primers and a heat-stable DNA polymerase.

14. What are primers? Why is a primer added?

Primers are small pieces of DNA that bind to specific sequences. In the case of the experiment,
the primers are added to bind the 16S rDNA region to initiate the replication process.

15. Once the primers bind, what occurs next?

Once the primers bind, the final step, known as extend, is to allow the DNA polymerase to extend
the copy DNA strand by raising the temperature to 70 degrees Celsius for 45 seconds.

16. What does "highly conserved" mean?

Highly conserved means that some parts of a bacterial species’ gene are very similar among
different species.
17. Why are highly conserved regions important in this lab?
Highly conserved regions are important because universal primers bind to them so that they
canbe used to copy DNA from a variety of bacteria species.

18. What does "highly variable" mean?

Highly variable refers to the regions of a gene that differ between species.

19. Why are highly variable regions important in this lab?

The highly variable regions of species are used for identification.

20. What is missing in the negative control tube?

DNA is missing from the negative control tube which only contains sterile deionized water.

21. What is present in the positive control tube that is not in the negative control tube?
The positive control DNA is contained in the positive control tube which the negative control tube
lacks. Now run the PCR. Be sure to watch the virtual lab animation before proceeding to the
questions.

22. List each step of a PCR cycle, the temperature, and the duration (time).
a. Melt: heated at 95 degrees Celsius for 30 seconds
b. Anneal: cooled to 60 degrees Celsius for 30 seconds
c. Extend: temperature is raised to 72 degrees Celsius for 45 seconds

23. Describe what happens during each of the steps in one or two sentences.
a. During the Melt period, the PCR reaction mixture sample is heated for 30 seconds at 95
degrees Celsius. This is to allow the DNA to separate the double helix chains.
b. At such high temperatures, the primers cannot bind to the DNA strands and so the sample is
cooled. Cooled now, the primers bind or anneal to the single-stranded DNA
c. The last phase is known as extend. During this phase, the temperature is raised to 70 degrees
Celsius for 45 seconds to allow the DNA polymerase to extend the copy DNA strand

24. After eight cycles, how many copies of the desired DNA have been synthesized?
After eight cycles, 240 copies of desired DNA have been synthesized.

25. After 30 cycles?

After thirty cycles, approximately 1 million copies of desired DNA have been synthesized.
PART 3: PCR PURIFICATION

26. Approximately how long is the 16s rDNA (bp)?

The 16S rDNA IS ABOUT 1500 base pairs (bp) long.

27. Why would it be useful to run an electrophoresis gel at this point?

It would be useful to run an electrophoresis gel at this point to confirm that the PCR reaction worked.

28. If you were to run a gel, it would have three lanes. What would each lane contain, and what would
you see in each lane after running the gel?
a. Negative control: this would contain water and should have no product unless the water was
contaminated
b. Positive control: this would contain PCR product of a known DNA sequence which would serve to make
sure the PCR worked
c. Last Lane: which contains the sample

29. The gel is not run in this virtual lab. In order to purify the PCR product, you use a micro concentrator
column. (Proceed through the virtual lab.) What should the final collection tube contain?
The final collection tube should contain many pieces of 1500 bp-long 16S rDNA with a small amount of
longer DNA strands which serve as contaminants.
PART 4: SEQUENCING PREPARATION Click on "Learn about cycle sequencing before proceeding."

30. Read the first two paragraphs and list the steps in cycle sequencing in the space provided
a Use a thermocycler to create many copies of the target DNA,
b Terminating the replication process at random places in the process to ensure the copies are
different lengths comes next, usually as a result of creating the copies
c Use electrophoresis gel to separate the pieces based on sized Compile the sequence for
complementary DNA

31. What do the green and blue tubes contain?

Describe the “sequencing brew” to which you added your purified PCR.The green and blue tubes
both sequencing brew which consist of buffers, primers, DNA polymerases, nucleotides and
fluorescence-tagged terminators in suitable proportions.

32. The purpose of the second PCR is not to create identical copies like the first PCR you ran. What is
thepurpose of this PCR?
The aim of the second PCR is to produce many copies of various lengths as opposed to identical
copies of DNA.

33. Where do scientists obtain primers to be used in PCR and in this technique?
Scientists obtain primers used in PCR and the sequencing brew technique through commercial
sources
PART 5: DNA SEQUENCING

34. What is the final PCR product, the stuff contained in your 12 tubes?
The final PCR product contained in my 12 tubes is a mix of DNA pieces of various lengths, starting
with the same primer but ending with a different nucleotide tagged with a fluorescent marker.

35. What is the purpose of gel electrophoresis?

The purpose of gel electrophoresis is a method to separate molecules based on size difference.

36. How do DNA molecules move in relation to charge? Why?

DNA molecules are negatively charged and as such tend to move through the tube toward the
positively charged syringe end. Smaller pieces move faster than the large ones.

37. What is the purpose of the laser beam in determining a DNA sequence?
The laser beam’s purpose in determining a DNA sequence is to excite the fluorescent markers
allowing optical detectors to detect the fluorescence color.
PART 6: DNA SEQUENCE ANALYSIS Click on "Learn about the science behind sequence matching."

38. What is the ultimate goal of the sequence matching analysis?

The ultimate goal of the sequence matching analysis is to determine whether the new sequence
of interest bears a significant degree of similarity to another known sequence

39. What is "homology"?

Homology is the similarity between two sequences.

40. What is BLAST and how is it used?

BLAST is the Basic Local Alignment Search Tool used for comparing sequence information in
proteins or DNA by utilizing all available public sequence databases.

41. What’s a major assumption when drawing evolutionary relationships between organisms based on
DNA sequences?
The major assumption when drawing evolutionary relationships between organisms based on
DNA sequences Is that the number of positions that differ in the nucleotide sequence is
proportional to the time elapsed since the two species formed their own lines of descent from a
common predecessor.

42. Explain what the "Score (bits)" means on an actual BLAST search result.
The Score on a BLAST search result is the numerical score assigned by BLAST while the bits are
the measure of information.

43. What does an E-value of 3 or less represent?

Am E-value of 3 or less represents a meaningful match that is not due to chance that is considered
an acceptable match.

44. What is the scientific name of the bacterium you sequenced?

The scientific name for the bacterium I sequenced is Bartonella ensilage.

45. Write a brief description of this bacterium in the space provided.

Bartonella henselae are pathogenic to humans. There are various species and they are usually
transmitted via a vector or from an animal reservoir, such as cats in the case of cat scratch
disease. Symptoms of the fever usual manifest in swollen lymph glands, potential skin lesions,
potentially fever and fatigue amongst other symptoms. After completing Sample A, perform DNA
sequence analysis on three of the other five samples.
46. Write in the letter of the samples you choose, the scientific name of the bacterium (after doing a
BLAST search), and a brief description of each.

Sample Letter Bacteria Scientific Name Brief Description

C Pseudomonas aeruginosa Commonly found in soil, water
and other natural
environmentsAdept at growing
in unusual sources like soap
residue When a person is
healthy, there’s not usually
much of a health threat
D Salmonella typhimurium Commonly found in the
intestinal tract of mammals,
birds and reptilesThe most
severe illness caused by these
bacteria is typhoid feverA
common source for these
bacteria is uncooked eggs
F Yersina enterocolitica These bacteria caused the
plagueCarried by urban and
rural rats, via their fleas which
will even settle for a human
hostCan potentially be
contagious by air as well