JMU BIO 150 Lab Midterm

Where would the following best fit in this flowchart of the “process of science”?

Your instructor provides you with edits and suggestions on a draft of your DNA barcoding project proposal

Community Analysis and Feedback

What type of study is this?

You want to test if a new soil sampling technique will improve the recovery of microbial DNA for PCR. To do this, you sample soil from 10 different locations that have previously given poor results with your current sampling method. You first collect soil samples from all 10 locations using the OLD sampling technique. Then, you collect soil from the same 10 locations using the NEW sampling technique. Next, you extract DNA from all the soil samples in exactly the same way (same kit, protocol, etc.). Afterward, you amplify the extracted DNA using the same primers, PCR conditions, and master mix for all samples. Finally, you run all the PCR products on an agarose gel to observe which samples amplified successfully.

Experimental

What is the independent, dependent, control and treatment group in the study?

Independent: Soil sampling technique

Dependent: PCR amplification success

Treatment: The new Soil sampling technique

Control: The old soil sampling technique

The results of this experiment:

All 10 samples extracted using the NEW technique SUCCESSFULLY amplified.

All 10 samples extracted using the OLD technique FAILED to amplify.

Given this result and assuming the methodology used in this study was sound (aka no user error or design flaws), which of the following conclusions is most appropriate?

A.) Although the new extraction technique would better, we cannot establish that the new technique caused the success (correlation is not causation)

B.) This study established/supports causation: The new extraction technique caused the increased PCR success.

C.) This study neither supports nor refutes that the new extraction technique caused an increase in PCR success

B.(Because all other variables were held constant. Assuming no user error or non-apparent flaws in the experiment design, the only logical explanation for the enhanced success of the samples in the treatment group is the treatment itself)

Which of the following are reasons specific genomic regions like rbcl, CO1, and ITS were chosen to use for DNA barcoding? (select all correct answers)

A.) There are many copes for these regions in each cell

B.) These regions have high intraspecific variation and low interspecies

C.) These regions have high interspecies variation and low intraspecies variation

D.) These regions have highly conserved flanking sequences so a single primer set can be used on many species

F.) These regions have highly variable flanking sequences, which can be used to tell closely related species apart

A, C and D

True or False: Primer dimers form when PCR primers bind to each other instead of the DNA template because they have complementary sequences

True (Primer dimers form when primers bind to each other instead of the target DNA bc that have complementary sequences. This can interfere with PCR by reducing the availability of primers for proper DNA amplification)

Another student group selected four samples of sushi for their project. The nanodrop showed that all four samples had DNA concentrations of ~100ng?ul and A260:A280 of exactly 1.8, so they proceeded to PCR and got the following gel. (Of teh four options below, choose the one that BEST explains these results)

A.) Their PCR primers were better matches for the DNA in samples A and B than samples C and D

B.)Their PCR primers were better matches for the DNA in samples C and D than samples A and B

C.) Their DNA concentration was below the optimum range for PCR, so there was not enough template to amplify

D.) Their samples likely had significant protein contamination that inhibited their PCR

A. (Because there are slight amplification in samples A and B and not C and D)

Another student group collected four vertebrate animal samples and amplified them using the vert CO1 primers. They then ran their PCR products out on a gel and got the following image. What's wrong with this gel?

A.) The streaking in sample JF04 indicates that an excess of primers were added to the reaction

B.)The size of these PCR amplicons is outside of the 500-800bp range we would expect for a barcoding project

(C) The band in the positive control lane indicates the presence of foreign DNA contamination in the master mix

(D) The band in the negative control lane indicates the presence of foreign DNA contamination in the master mix

(F) Nothing is wrong, this gel looks ideal and they should proceed to sequencing

D.

After appropriately diluting their samples, Sandra, Chris, and Solomon went ahead and PCR amplified their DNA extracts. They then ran a gel of their PCR products. Lanes 1-3 are their unknown samples, Pos indicates the positive control, and neg indicates the negative control. Which of the following statements is/are true about their gel?

A.) All of their unknown samples amplified

B.) One or more of their primers are annealing to non-target regions of the genome (there is non-specific binding)

C.) Their controls show that the master mix was contaminated by foreign DNA

D.) The streaking in sample 3 between 1kb and 1.5kb is likely due to the formation of primer dimers

E.) The positive control did not amplify correctly

F.) The amplicon size for the main band in lane 1 matches their predictions from ApE (~600bp)

A, B, E and F

(The streaking in lane 3 is not due to primer dimers. Primer dimers are very small DNA fragments and are never as large as 1kb. This streaking is likely due to too much template DNA in the reaction, though it is not bad enough that this PCR would need to be repeated.)

James used ApE to analyse his primers. The blue highlighted region also includes both primer binding sites and all of the bases in between,

How many bp long would he expect his PCR product be? (provide a numerical answer, do not include bp)

599

Sandra used ApE to determine if her primers were a good match for her expected fungal species. The anticipated primer binding sites are highlited in blue on the ApE window below. She found that the forward primer matched with zero mismatches. What was the sequence of Sandra's forward primer? (do not include 5' and 3' in your answer)

TCCGTAGGTGAACCTGCGG

You make a master mix spreadsheet that will automatically calculate the appropriate volumes of each ingredient.

Which of the following is the most appropriate equation to "adaptively" calculate the total volume of PCR reactants (aka cell B17)?

(NOTE: adaptively means you don't have to recalculate the answer by hand each time. The spreadsheet does it automatically. If you're still not sure what this means, please ask your instructor for clarification).

A.) =B12*B5

B.) =15*9

C.) =B12*9

D.) =9*(30-15)

A.

Magret knows for sure that his DNA concentration is too high, so he plans to dilute his sample to 50ng/ul before proceeding to PCR. If Solomon wants to make 100ul of diluted extract, how many microliters (ul) of his sample should he add to his dilution?

(provide your answer rounded to one decimal place and do not include units. For example, if he needs to add 50.66667ul, enter 50.7).

7.6 ( The formula for calculating a dilution is: (Starting concentration)(starting volume)=(final concentration)(Final volume)

(The starting concentration is the nanodrop readout : 657.4ng/ul

The starting volume is unknown

The final concentration is 50ng/ul

The final volume is 100ul

So... (657.4)(x) = (50) (100)= 7.6uL

How many microliters (ul) of water should Solomon (from the question above) add to his dilution to achieve the desired concentration? (provide your answer rounded to one decimal place and do not include units)

92.4 (bc 100uL-7.6uL=92.4)

Penoy also extracted a sample. When he used the nanodrop to quality control his extract, he got the following results. What should Chris do about his DNA extract?

A.)He should proceed directly to PCR. Everything looks great.

B.) He should repeat his DNA extraction, because his DNA concentration is too low

C.) He should dilute his DNA extract, because his A260:A230 is low

D.) He should dilute his DNA extract because his A260:A280 is below 1.8 indicating significant salt contamination

B. (A DNA concentration of 15.8ng/ul is well below our desired range, and this sample may also contain significant PCR inhibitors due to a low A260:230.)

Jessica extracted DNA from a mold sample, and then ran her extract through the nanodrop and got the following result. Which of the following statements are TRUE about Sandra's extract?

A.) The DNA concentration is in optimal range for PCR

B.) The A260:A280 is in an acceptable range to proceed to PCR

C.) The A260:A230 is in an acceptable range to proceed to PCR

D.) This sample should be diluted because the DNA concentration is too high

E.) This sample should be diluted because the purity ratios are too low

F.) This sample should not be diluted, it is ready for PCR

B, C and D (Both of our purity ratios are well above 1.4 indicating a very pure sample. The DNA concentration is higher than optimal for PCR, but that's a problem we can easily solve with dilution. This sample should be diluted to 50-100ng/ul before proceeding to PCR.)

In lab, we accessed NCBI GenBank for our primer mapping activity. What is GenBank?

A.) Genbank is one of the world's largest repositories of DNA sequence information

B.) GenBank is a piece of free software that calculates the ideal Taq polymerase concentration for a given PCR reaction

C.) GenBank is a piece of free software that tells you whether your PCR is going to be successful

D.) GenBank is a website that calculates DNA concentrations from Nanodrop data

A. (GenBank is one of the world's largest DNA sequence databases! This is the website you went to to find a sequence of your organism of interest for the primer mapping activity.

GenBank does not calculate Taq concentrations, or deal with Nanodrop data, and there's no software in the world that can actually tell you if a given PCR will be successful )

5'CAATGCGTGGGTCATTATCCAAAGACTTATG-3'

You have developed primers against the the underlined regions of the DNA sequence above. Using these primers, what would the length of the PCR amplicon produced using these primers?

A.) 31bp

B.) 8bp

C.) 16bp

D.) 15bp

A. (The expected PCR amplicon length is the length of the fwd primer + the length of your reverse primer plus the number of bp between the two binding sites. In this case the primers are each 8bp long and there are 15bp between the two binding sites so:

8+15+ 8 = 31bp)

5'CAATGCGTGGGTCATTATCCAAAGACTTATG-3'

You want to develop primers that would amplify the entire sequence listed above. Each of your primers will be 8 bp in length.

Write the sequence of the forward primer (from 5' to 3') in the space provided below.

(Be sure to pay close attention to labeling of the 5’ and 3’ ends. Do not include "5'" or "3'" in your answer. Use capital letters with no spaces in between: EX. GGCCTAC).

CAATGCGT (PRACTICAL NOTE FOR ANSWERING THIS KIND OF QUESTION : Your forward primer will be identical to the template sequence and your reverse primer will be the reverse complement of the template sequence you are given, written 5' to 3'.)

5'CAATGCGTGGGTCATTATCCAAAGACTTATG-3'

You want to develop primers that would amplify the entire sequence listed above. Each of your primers will be 8 bp in length.

Write the sequence of the reverse primer (from 5' to 3') in the space provided below.

(Be sure to pay close attention to labeling of the 5’ and 3’ ends. Do not include "5'" or "3'" in your answer. Use capital letters with no spaces in between: EX. GGCCTAC).

CATAAGTC

Which of the following statements is/are true about the annealing of primers to a DNA template? (select all that apply)

A.) None of these statements are true

B.) Individual primer molecules can be reused through PCR cycles and never get "used up". This is why you can run so many PCR cycles without running out of primers.

C.) PCR primers are usually ~25bp long, and anneal to a ~25bp region of the template

D.)The two strands of the template DNA must first be separated by heating before the primers can anneal

E.) Primers can only bind if they are a 100% complementary to the DNA template (there are no mismatches)

C and D (C is correct because it is a long enough sequence that it can be found in only a single region of the genome, thus preventing non-specific binding. When primers anneal they form complementary basepairs with the template. This means that one primer nucleotide binds to one template nucleotide, so the length of the binding site will be identical to the length of the primer.)

(D is correct because : template strands must be separated by heating before primers can bind. This occurs during the denaturation phase of PCR.)

Which of the following statements is/are correct regarding DNA template concentration and purity? (select all that apply)

A.) PCR reactions work best with lots of template DNA. In fact, the more template the better!

B.) PCR reactions work best with very low concentrations of template DNA. A single template DNA strand would be ideal because it would produce a very pure amplicon.

C.) A low A260:A280 ratio suggests that your sample contains very little salt contamination, so it is likely to amplify successfully

D.) An A260:A230 ratio of approximately 2 suggests that you have very pure DNA sample, so it is likely to amplify successfully

D (PCRs work best with intermediate concentrations of DNA, somewhere around 100ng/ul. Too much or too little DNA can both cause PCRs to fail, so neither A or B are true.

The A260:A280 ratio tells you about protein contamination, not salt contamination, and low ratios indicate more contamination, not less. A low A260:280 ratio would indicate significant protein contamination, which could inhibit your PCR reaction.

A260:A230 tells you about contamination by substances that absorb light around 230nm, including things like salts and carbohydrates. Pure DNA would have a value of 2-2.2 for this ratio, so this answer is correct.)

True or false: In a PCR reaction denaturation usually occurs immediately after extension.

HINT: A standard PCR run goes through ~30 cycles.

True (because PCR goes through repeated cycles. Each individual cycle goes denaturation-annealing-extension, but because the cycles repeat ~30 times in a PCR run for every cycle except the very first one, denaturation does occur after extension.)

Which of the following statements is/are true about the structure of DNA? (select all that apply)

A.) DNA is always double stranded throughout the PCR process

B.) Adenine and thymine form complementary base pairs

C.)Adenine and guanine form complementary base pairs

D.) Each strand of DNA has a 5' and 3' end

E.) In double stranded DNA, DNA strands are arranged in a parallel arrangement

B and D

(DNA strands are not arranged in parallel, they are arranged anti-parallel, with the 3' end of one strand and the 5' end of the other at the same end of the double stranded molecule.)