Bacterial Identification PCR and DNA Sequencing Lab Report Fall 2023 (1)

pdf

School

American River College *

*We aren’t endorsed by this school

Course

310

Subject

Biology

Date

Feb 20, 2024

Type

pdf

Pages

Uploaded by ChiefPrairieDog3572

Introduction 1 The purpose of this virtual lab is to familiarize you with the science and techniques (PCR and DNA sequencing) used to identify different types of bacteria based on their DNA sequences. Remember that not all bacteria are harmful to people. However, approximately 40% of bacteria are pathogenic or disease-causing bacteria. Before treating a pathogenic bacterium, it is important to know what kind of bacteria is present. While there are a variety of methods to identify bacteria, modern biotechnology techniques have made identification faster and more precise. Overview 1. Go to the following link: https://www.biointeractive.org/classroom-resources/bacterial-identification-virtual-lab 2. Once you are at the site, right underneath the title of the lab, on the left side of the screen there is small green button "start interactive". Click that button to launch the virtual lab activity. 3. Read the introduction (under the Introduction tab) and answer the following questions: a. What are the four basic steps involved in this bacterial identification lab? 1. Prepare a sample from a patient and isolate whole bacterial DNA. 2. Make many copies of the desired piece of DNA. 3. Sequence the DNA. 4. Analyze the sequence and identify the bacteria. b. What is "16S rDNA," and how is it used to identify species of bacteria? 16S rDNA sequencing is a powerful tool for identifying and classifying bacterial species. The identification relies on matching the sequence from your sample against a database of all known 16S rDNA sequences. 1 Adapted from Howard Hughes Medical Institute (hhmi) BioInteractive. (2018). Bacterial Identification Virtual Lab. https://www.hhmi.org/biointeractive (accessed 11/19/2018).

c. Review: What does a ribosome do? Are they found in prokaryotic cells, eukaryotic cells, or both? A ribosome is a cellular structure responsible for protein synthesis.Ribosomes are found in both prokaryotic and eukaryotic cells. d. What is Gel Electrophoresis used for? Used for separating and analyzing molecules such as DNA or proteins. Conduct Virtual Lab 1. Click the window on the left-hand side of the screen to “enter” the lab. 2. At the bottom of the lab screen you will see the six parts for completing this activity. Each step will have associated written information in the “notebook” tab on the right-hand side of the screen. Part 1 - Sample Preparation 3. Click on the Notebook tab. Read “Part 1 Sample Preparation” and answer the following questions: As the pathology lab technician, what is your task in this virtual lab? Your task is to identify a bacterial sample received from a clinician. Once you have grown several bacterial colonies, what is the first step in extracting the bacterial DNA? The first step is to pick up a single colony and drop it into a microcentrifuge tube. How long does this step take? Several hours. What is the purpose of the hot bath step? The purpose of heating a sample in a water bath at 100°C is to denature the enzymes present in the sample.

The centrifuge helps separate out the cellular debris (which we don’t want) from the DNA (which we do want). Where is the DNA found – in the pellet or the supernatant? Supernatant. 4. Enter the lab on the left-hand side. Follow the prompts and answer the following questions as you go: What is the wire ring for? For scooping up bacteria. Part 2 - PCR Amplification 5. In the notebook tab area, click on “What is PCR?” and answer the following questions: What does PCR stand for and what is its purpose? Polymerase Chain reaction is a technique that allows many copies of DNA to be made. What is the purpose of the heat in PCR? The purpose of heating is to make millions and billions of copies of DNA. What enzyme makes the new copies of the DNA? PCR. Why are the chemoautotrophic bacteria that live in deep sea hydrothermal vents important to modern PCR? The DNA polymerases remain functional at such temperatures that would have denatured an ordinary creature's DNA polymerases. 6. Click on “Back to Part 2” and answer these questions: What is in the PCR Master Mix?

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

Large quantities of the four nucleotides adenine, cytosine, guanine, and thymine; large quantities of oligonucleotide DNA primers that bind the 16S rDNA region to initiate the replication process. What are primers? Why is a primer added? A primer is a short nucleic acid sequence that provides a starting point for DNA synthesis.Primers are being added DNA because they serve as the starting point for DNA synthesis. What does it mean for a part of a gene to be "highly conserved"? How do these regions help us? It means they can be used to copy DNA from a variety of bacteria. What does it mean for a part of a gene to be "highly variable"? How do these regions help us? The differences between species and help for identification. What is your positive control? DNA. What is your negative control? Sterile deionized water. Describe what happens during each of the three steps of a single PCR cycle. Adding buffer solution, adding PCR product, adding buffer solution to the column.

How long does a single PCR cycle take? One minute 7. Now run the PCR. Watch the virtual lab animation before proceeding to the questions. After eight cycles, how many copies of the desired DNA have been synthesized? After 30 cycles? Eight cycles = 240 Thirty cycles = 1,073,741,764 Part 3 - PCR Purification 8. Read the notebook for this part and answer the following questions: Approximately how long is the 16s rDNA (bp)? 1500. What method will you use in this virtual lab to purify your DNA sample? Gel. 9. Now run the PCR purification. Part 4 - Sequencing Prep 10. Read the notebook for this part and answer the following questions: How is each of the green and blue tubes different for this step? Each green and blue tube contains a " sequencing brew " consisting of buffers , primers , DNA polymerases , nucleotides , and fluorescence - tagged terminators in suitable proportions .

How is the purpose of the PCR cycle sequencing step different from our original PCR amplification step? It’s possible to use a single primer in PCR cycle sequencing, but it is not feasible for long sequences. With multiple primers and overlapping stretches of DNA are sequenced to obtain the complete sequence. 11. Now run the PCR cycle sequencing. Are all the resultant DNA pieces the same length? NO. What do the colored ends represent? Primer or polymerase. Part 5 – DNA Sequencing 12. Read the notebook for this part and answer the following questions: What does gel electrophoresis do? A method to separate molecules based on differences in sizes. Is DNA positively or negatively charged? Negative charged. Which would move faster: 1500 base pair piece of DNA or a 400 base pair piece of DNA? Explain. Small pieces of DNA that have a fluorescent tag attached to it is the primer. This DNA fragment will travel faster than the other ones. By reading the color, we determine that the first nucleotide beyond the primer sequence is thymidine . Does it make sense that computers are an important part of compiling all this information?

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

Yes, for collecting information. 13. Now run the DNA sequencing. As the sequencer is running, is the order of bases the same for each tube it runs? It varies. Part 6 – Sequencing Analysis 14. DO NOT CLICK ON THE BACTERIA NAMES IN YOUR VIRTUAL LAB YET! Go to the notebook section, click on “Learn about the science behind sequence matching”, and answer the following questions: What is the ultimate goal of the sequence matching analysis? The ultimate goal of this analysis is to determine whether the new sequence has a significant degree of similarity to another known sequence . What is "homology"? Based on the same degree of similarities. What is BLAST and what is it used for? Basic Local Alignment Search Tool. 15. Go back to Part 6 and follow Steps 1-5 to identify your sample. Note: The BLAST search can sometimes take a full minute or two. When the results appear, scroll down below the color key to look at the “sequences producing significant alignments.” Take a screenshot of your blast results that also shows the date on your computer and insert it here. 16. Return to the virtual lab and select the name of the bacterium you sequenced.

What is its scientific name? Bartonella henselae. Write three facts about this type of bacteria. 17. Click on the Samples tab. Perform DNA sequence analysis on three of the other five samples and record your results below. Sample Letter Scientific Name 3 Facts

Related Documents

ThompsonWeek7Lab.docx

Human Structure StudyGuide 2.docx

Lab # 5.docx

Homework Week 1-katecoffman-dr.pineda.docx

Homework Week 1 2.docx

Photosynthesis Lab Report.docx

Mod_1_Handout & Summary_Activity.docx

Practice_Exam_1.pdf

Bio173 Plant data worksheet.docx

Module 3_ Macromolecules_ Fall_2023.docx

Chapter 3 study questions.docx

A3_Toder_Meghan.docx

Recommended textbooks for you

Biomedical Instrumentation Systems

Chemistry

ISBN:9781133478294

Author:Chatterjee

Publisher:Cengage

Human Heredity: Principles and Issues (MindTap Co...

Biology

ISBN:9781305251052

Author:Michael Cummings

Publisher:Cengage Learning

Biology 2e

Biology

ISBN:9781947172517

Author:Matthew Douglas, Jung Choi, Mary Ann Clark

Publisher:OpenStax

Biology Today and Tomorrow without Physiology (Mi...

Biology

ISBN:9781305117396

Author:Cecie Starr, Christine Evers, Lisa Starr

Publisher:Cengage Learning

Biology (MindTap Course List)

Biology

ISBN:9781337392938

Author:Eldra Solomon, Charles Martin, Diana W. Martin, Linda R. Berg

Publisher:Cengage Learning

SEE MORE TEXTBOOKS

Recommended textbooks for you

Biomedical Instrumentation Systems
Chemistry
ISBN:9781133478294
Author:Chatterjee
Publisher:Cengage
Human Heredity: Principles and Issues (MindTap Co...
Biology
ISBN:9781305251052
Author:Michael Cummings
Publisher:Cengage Learning
Biology 2e
Biology
ISBN:9781947172517
Author:Matthew Douglas, Jung Choi, Mary Ann Clark
Publisher:OpenStax
Biology Today and Tomorrow without Physiology (Mi...
Biology
ISBN:9781305117396
Author:Cecie Starr, Christine Evers, Lisa Starr
Publisher:Cengage Learning
Biology (MindTap Course List)
Biology
ISBN:9781337392938
Author:Eldra Solomon, Charles Martin, Diana W. Martin, Linda R. Berg
Publisher:Cengage Learning