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)?

DNA fingerprinting webquest

DNA DETECTIVE WORK

Unless you have a twin, no one in the world has a DNA sequence identical to yours. Although 99 percent of the DNA is the same in all humans, certain segments vary widely. Differences in these segments are detected through DNA fingerprinting. A small amount of tissue, such as blood, hair, or semen, is all that is needed to create a DNA fingerprint. The sample is cut up using enzymes and the segments are separated by size through gel electrophoresis. DNA is made visible either with radioactive probes or by staining. This reveals a pattern of bars: the DNA fingerprint. If the two DNA fingerprints match, they probably came from the same person. If they don't match, they certainly came from different individuals. In recent years, a number of people convicted of crimes have been exonerated based on DNA evidence.

Activity

In this exercise, you will learn the basics of DNA fingerprinting and consider the use of DNA in criminal investigations.

Part 1.

Use your browser to go to NOVA’s web site about “Killers Trail,”  the story behind the man who inspired the Fugitive TV series and later the movie version starring Harrison Ford:

http://www.pbs.org/wgbh/nova/sheppard/

Click on the “Chronology of a Murder” section and read about the events that led up to the murder trial of Dr. Sam Sheppard.

1. In your opinion, what role (if any) did newspaper stories and editorials have in the outcome of the original trial of Dr. Sam Sheppard?

yes i do because the writers of the stores were trying to get the dr. convicted because they were putting lies in the stories they wrote.

Go back to the Killers Trail homepage and select ”Create a DNA Fingerprint.”

Read about the crime and the suspects then go on to part 2. Answer the following questions about the technique as you go through the simulation:

2. What is the function of the restriction enzymes in DNA fingerprinting?

the restriction enzymes work like scissors cutting the long dna molecules at diffrent locations

3. What is the function of  the agarose gel electrophoresis step?

it will act like a molecular strainer 

4. Why is a nylon membrane used to blot the DNA?

because it hard to pick up the jell so the dna is trnsfred the the nylon membrane so it is easy to pick up and work with

5. What does a dark spot on the X-ray film indicate?

it indicates were the DNA mixture is

Part 2.

Use your browser to go to Frontline's "What Jennifer Saw" at

http://www.pbs.org/wgbh/pages/frontline/shows/dna/.

The material on this site is about a man convicted of rape but later exonerated by DNA evidence. To read a summary of the case, choose the link to Ronald Cotton's wrongful conviction, then choose "Summary of Cotton's Case."

In the interviews section, read the interviews with DNA expert Peter Neufeld and lawyer Barry Scheck.

Answer the following questions:

6. What evidence was initially used to convict Cotton?

a photo , a police identification line up and, a flashlight similar to the one at the crime and the rubber of his shoes.

7. What did the DNA evidence show?

the DNA evidence showed the it was the man who emited to the crime

8. How could DNA fingerprinting be used to prevent a false conviction if a case like this was being tried today?

it could because it would go throw the database of dnas and find the right match.

9. What percentage of convicts are unjustly convicted of sexual assault cases, according to Neufeld and Scheck?

25% of people ar unjustly convicted of sexual assault cases.

10. The O.J. Simpson trial was one of the most visible trials that attempted to use DNA evidence.  In the end, the DNA evidence was not satisfying to the jury, who acquitted Simpson.  What do Neufeld and Scheck believe about the impact of the O.J. Simpson trial on the use of DNA evidence?

 is it highlights on the one hand the tremendous potential of this technology that it is a science that can be the equivalent of the videotape of the commission of the crime. Yet, on the other hand, it points out that this is not a law of science that we're dealing with, such physics such as the law of gravity.

Instead, we're talking about an applied science, an applied technology and whenever people get involved in the application of science is much opportunity for mistake, error and for much worse. And so we have to be very rigorous in the kinds controls that we exert when we utilize this tremendously powerful new technology to make sure that it's used wisely and cautiously.

DNA Sequence Comparisons Between Species: Molecular Evolution

EXPLORING MOLECULAR EVOLUTION

STUDENT WORKSHEET

Results of your pairwise alignment comparing the beta globin gene in humans and in chimps:

1. Data about the alignment can be found below the blue/black alignment chart. How many base pairs (bp) are there in the beta globin gene for:
1. The chimp? 600

2. The human? 626

2. A blue asterisk indicates that the nucleotides (bp) in both sequences are the same, we say they are conserved. What percentage of the beta globin sequence is conserved in chimps and humans? (Don’t include the insertion at the beginning of the human gene). This percentage is often reported as a similarity “score” below the alignment.

11333

3. Would you expect the protein structure to be highly similar or markedly different in the chimp and the human? Explain.

i think i would be highly similar because the chimps have the closet dna structure the the humans.

RETURN TO BIOLOGY WORKBENCH INSTRUCTIONS

Results of your pairwise alignment comparing the beta globin gene in humans and in chickens:

1. What is the percentage of sequence conservation between the beta globin gene in chickens and humans?

2. Looking at the two pairwise alignments you have performed, would you expect the beta globin protein found in humans to be more similar to that found in chickens or that found in chimps? Explain.

it is more similar the one found in chimps because it think it is because we have a dna sequence most closely to the ones of chimps

3. Do the results achieved by running these alignments support the results on evolutionary relationships determined by scientists using anatomical homology (similarities)? Explain.  

i think that i might help their theories but man never came from ape because we do not have the same dna sequence.

RETURN TO BIOLOGY WORKBENCH INSTRUCTIONS

Results of your multiple sequence alignment comparing the beta globin gene in a variety of animal species:

1. Examine the Unrooted Tree produced.  

Record the species at the end of each branch on the unrooted tree shown below.

2. Based on the information in the unrooted tree:

1. Which two species appear to be most closely related to each other? Explain your choice.

chimp and human because the had the closest of the beta globin the was the same

2. Which two species seem to be the least closely related to each other? Explain your choice.

chicken and human because the beta globin was far apart from each other

3. Comparative evolutionary distance between species is indicated by the length of the clades they are on. Give the comparative evolutionary distance (by percentage similarity “score”) between:

1. The mouse and human

2. The wallaby and the human

3. The chimp and the human

Comment on the significance of these results given your knowledge of mammalian groups.

RETURN TO BIOLOGY WORKBENCH INSTRUCTIONS

Results of your Rooted Phylogenetic Tree:

1. Examine your Rooted Phylogenetic Tree and record the species at the end of each branch.  

2. Based on this tree diagram, which species is/are most closely related to:

1. The goldfish: chicken

2. The mouse: human, chimp

3. Homology is a term used to refer to a feature in two or more species that is similar because of descent; it evolved from the same feature in the last common ancestor of the species. Hence, similarity in DNA or protein sequences between individuals of the same species or among different species is referred to as sequence homology. Which two species in the tree above share greatest homology with respect to the beta globin gene?

the human and the chimp

4. A node is a branch point representing a divergence event from a common ancestor. Which two species have the most ancestral nodes (divergence events) in the tree above? Explain your answer giving the number of nodes leading to these species.

chimps and humans and the nodes are 3

5. Looking at the phylogenetic tree above, which two organisms:

1. Diverged from their common ancestor most recently?

the wallaby

2. Diverged from their common ancestor least recently?

chimp human and the mouse

6. Draw a modified phylogenetic tree to show how the tree above might change if the beta globin gene for a kangaroo was added to the multiple sequence alignment.

7. It is important to understand that the phylogenetic trees you generated using bioinformatics tools are based on sequence data alone. While sequence relatedness can be very powerful as a predictor of the relatedness of species, other methods must be used in addition to sequence homology, to determine evolutionary relationships. Briefly describe 3 other methods that you think might be used to determine evolutionary relationships.

• . Fossil evidence (they see which fossils are more structurally related)
• Embryology (looking at the embryos of developing young. Pretty much every mammalian embryo looks exactly the same at some point. Scientists study how long in development two embryos remain similar to compare evolutionary relationships)
• Anatomy (which creatures and body parts look structurally similar? Even though fish and whales both have fins, if you study the anatomy, you realize that the whale actually descended from a wolf like creature)

Bacterial Transformation Lab

Overview

So for this lab we had four Plates.Each plate had 4 different plates with 4 different substances/mixture.

So we started the lab with 2 separate test tube. One is + and one is -

We then put a solution into both of the test tubes. In which we then placed in ice. We then grabbed bacteria from one of the plates onto both test tubes. We then place the plasmid onto the + and put both tubes back into the ice. We put both test tube into ice for about 10 minutes. Now after the 10 minutes of sitting in the ice we directly put it in 42 C in warm water for about 50 seconds. We then put it back into the ice for about 2 minutes. After we open both of the test tubes and put LB nutrient broth onto both test tubes. We then had to keep the test tubes at room temperature so we held the test tubes in our hands for 10 minutes. We then placê the + pglo test tube into the 3 test placê Lb/amp,Lb/amp/ara,Lb. On the - test tube Lb/amp we placed the -pglo test tube . We then grabed a sterile loop for each plate and we spread each of it around on each plate. We then stacked our plates and and labeled it and stuck it in the incubator and this was our results.

Add caption

This tray had regular food/plasma/spiked with Ameslan.

This tray had regular food,plasma/spiked with Ameslan/and spiked with Ara a sugar.














This tray just had regular food.All the plates have e-coil bacteria .

This tray had regular food/No plasmid and was spiked with Ameslan

Conclusion

The LB AMP Ara was the only plate that would glow while the others didnt.

The LB+,
LB/Amp+,
LB/Amp/ARA+ =had lots of growth

The LB/Amp had =no growth .

The Lb/AMP=0 bacterial colonizes 

The LB/Amp/ARA+=1000 colonizes

The Lb/Amp+=800 colonizes about the same amount but has a little bit less.

The LB+=40 colonizes

The Lb/AMP + got new dna because it had growth and got the new dna while the Lb/AMP - didnt because it didnt get the plasmid while the LB/AMP + did.

Lb/AMP/ARA got new dna because its glowing which shows that it had plasmid/and growth which shows that it glows.
The Lb + got the new dna because it had lots of growth and was still producing cells.