JMU BIO 150 Lab Final

tertiary literature

"third-hand" information that is typically written by non-experts, often lacks citations, often prone to generalizations and simplifications, and not typically peer reviewed

Which of the following can be very helpful in comparing levels of polymorphism a MUSCLE alignment?

% sequence similarity

The three main stages of the PCR process are usually repeated around 30 times over several hours. Approximately how many copies of the target region of the original DNA molecule are made during that time (assuming perfect amplification)?

1 billion (= 2^30)

A260:230 ratio, below which indicates potentially significant contamination

1.4

A260:280 ratio, below which indicates potentially significant contamination

1.4

ideal A260:280

1.8

You have 50 uL of DNA extract with a DNA concentration of 200 ng per uL. What is your Total Yield of DNA?

10,000 ng

1.23 mL (milliliters) = _______ µL (microliters)

1230 µL

ideal A260:230

2.2

salts and carbs: peak wavelength of light it absorbs

230nm

DNA: peak wavelength of light it absorbs

260nm

proteins: peak wavelength of light it absorbs

280nm

You want to develop primers for the blue and red regions of the following DNA template sequence: 5'-ATGCAATTGCAGTCGATTGCTCGATCGATCTTCGAAATTTCCCGAGCTTCGATCGC-3' Which of the following is the appropriate Reverse Primer sequence?

5'-GCGATCGAAGCTCG-3'

ideal DNA concentration

50-250 ng/uL

You want to develop primers for the blue and red regions of the following DNA template sequence: 5'-ATGCAATTGCAGTCGATTCGATACGATCTTCGAAATTTCCCGAGCTTCGATCGC-3' How many base pairs long is the PCR product generated by this primer set? (assume the primers only bind to the sequence above)

54 bp

What volume of PCR product goes into an individual sequencing tube?

8 ul

Which of the following would be a GOOD region of the genome to use for DNA barcoding?

A region that has lots of copies in the genome

This number can indicate the amount of salt or carbohydrate contaminants in your DNA extract

A260:230

Which of the following BEST describes the general structure of an introduction:

An upside down pyramid that starts with a hook to get the audience interested and all of the following information helps the reader to understand the specific aim of the study and its importance

Which of the following should you typically include in a discussion section? (check all that apply).

Any follow up questions you might logically ask based on your results How your results compare to what you expected to see An explanation of unexpected results

According to your readings from this week's lab, which of the following is/are question(s) that "science" can address. Note that not all answers must be about DNA barcoding to be correct. (check all that apply)

Are sushi restaurants selling cheap species of fish for more $$? What is the average number of burritos consumed by a JMU freshman? How many species of insects are in cow dung?

Which of the following is not a "SEQUENCE ASSEMBLY' substop in the DNA subway blue line?

BLASTN

Pat wants to use DNA barcoding to determine what % of pork sausage is mislabelled at Walmart vs. Local Butchers, but he can only collect 20 total meat samples. Which of the following is the best experimental design to test this (based on the principals of sample independence and balanced design)?

Barcode 10 sausages each from 10 different packages at walmart and 10 from 10 different packages/batches from the butcher

According to your lab manual, what are the 2 main things that citing your sources accomplishes? (check the two that apply)

Citing your sources establishes the source of facts that are stated in a paper and therefore the legitimacy (or illegitimacy) of these statements Citing your sources provides readers with an "in-road" to the literature surrounding a particular topic

CRAAP stands for?

Currency, Relevance, Authority, Accuracy, Purpose

Danielle wants to use DNA barcoding to determine the identity of the plants in her backyard. What kind of study is she conducting?

Descriptive

Why is 70% ethanol used in the plant DNA extraction protocol?

Ethanol helps wash away impurities from your DNA

Where would the following item best fit in this flowchart of the "process of science"? You conduct a web search to learn more about DNA barcoding and what kinds of questions it is being used to answer

Exploration and Discovery

Which of the following is the typical "entryway" into the process of science?

Exploration and Discovery

For the greatest accuracy in pipetting, one person should hold the pipettor while a second person holds the test tube the person is pipetting into or out of

False

Jessica extracted DNA from bee samples and used CO1 primers in her PCR mastermix to amplify the bee DNA. Ladder/marker, positive and negative controls all worked properly in her gel, but her sample did not amplify. In later ApE analysis, she found that these CO1 primers did not align anywhere in the genome of her bee species nor in the genomes of several closely related species of bees. Given this info. what is her best next move?

Get new primers that will anneal better

The primer used in Sanger sequencing

Has a nucleotide sequence complementary to the area that flanks the region you want to sequence

Which of the following is NOT a tip for successful pipetting?

Hold the pipettor horizontally after pulling a sample

Which of the following is/are true regarding the procedure to select BLAST hits for later analysis?

In general you should avoid selecting hits that are NOT ID'd to the species level (Ex. Uncultured Basidiomycota or Gomphidae or Tristus spp.) you should select the top ~4-5 taxonomically unique hits

What is the objective of a discussion section of a scientific research paper?

Interpret your results in the context of the specific question you set out to address

Why do we run our H2O control (aka negative control) from our PCR reactions on the gel?

It enables you to tell if your PCR was contaminated by DNA that was not from your sample

Which of the following are advantages of scholarly literature? (Choose all that apply)

It is transparent with sources of information It is detailed It is reliable

Why do scientists load DNA of known sizes (also called "marker" or "ladder") into the agarose gel?

It makes it easier to determine sizes of unknowns using comparison techniques

The final step in a Sanger DNA sequencing reaction is to run the DNA fragments on a gel. What purpose does this serve?

It separates DNA fragments generated during the sequencing reaction based on one-nucleotide differences in their size.

How can contamination be prevented in the laboratory?

Keeping test tubes and pipette tip boxes closed as much as possible. Using barrier pipette tips. Changing pipette tips between solutions, or after tips have touched DNA.

Which is a disadvantage of scholarly literature?

Less accessible to non-experts

Which of the following statements about gel electrophoresis is correct?

Migration distance is inversely proportional to the fragment size

Dideoxy (aka Sanger) DNA sequencing makes use of monomers called ddNTPs that stop DNA synthesis in predictable ways. This allows researchers to determine the sequence of bases present in a strand of DNA. The master mix of ingredients used in the DNA synthesis reactions of Sanger dideoxy sequencing include few ddNTPs relative to normal dNTPs (A's, T's, G's, and C's). What would be the most likely outcome of a Sanger dideoxy DNA synthesis reaction if ddNTPs were present in large numbers relative to the number of dNTPs (normal A's, T's, G's, and C's)? (NOTE: a daughter strand is a strand of DNA that is replicated from the original DNA template that is being sequenced)

Most daughter strands would be very short.

What is NCBI Genbank?

One of the world's largest DNA sequence Databases

What does peer-reviewed mean?

Other experts in the field have verified the integrity of the information

During PCR, DNA polymerase (or Taq polymerase) starts copying at

Primers attached to the end of the desired DNA sequence

Which of the following is NOT an item that belongs in the process of science flow chart?

Research

Which of the following statements is FALSE with regards to scholarly literature?

Scholarly literature is typically short in length

Which of the following is a major advantage of a subject specific database (like Scopus) over Google Scholar?

Scopus has better filters than Google Scholar to help narrow down your results

Electrophoresis is used to:

Separate DNA fragments Determine the size of DNA fragments Determine the presence of DNA fragments of certain size

Which of the following are examples of "Exploration and Discovery" (check all that apply)

Sharing data and ideas Making Observations Asking Questions Finding inspiration Exploring the literature

Typically it is appropriate to BEGIN a discussion section by:

Stating your main hypothesis/question, followed by whether or not your results supported your main hypothesis/addressed your question

What is the objective of a results section of a scientific research paper?

Summarize your findings using tables, graphs, and words

What goes in the green circle of this flow chart depicting the process of science?

Testing Ideas

Which of the following would typically be included in the results section of a scientific research paper? (check all that apply)

Text that describes the important trends/findings in your figures or tables (Example: We placed eight atmospheric monitoring stations within a 6-km2 area (Table 3)). Images or tables that summarize your data In text citations of your figures/tables (Ex. (Fig.1))

If no primers were included in your PCR the reaction would not work because:

The DNA polymerase would not amplify the specific region of DNA you want to be amplified

You want to see if a new chemical you have will help improve the success of PCR amplification of DNA from your samples. To test this you extract DNA from 10 samples that you know have worked in the past with the normal PCR chemicals you always use. Then you use two different PCR MASTER MIXES on the DNA extracts. In one PCR mix you use the NEW chemical. In the other PCR mix you use the old chemicals. Everything else is exactly the same. Then you run ALL the PCR products out on a gel to see which ones successfully amplified DNA. What is the control treatment in this study?

The original PCR mix with the old chemicals

The rate of migration of DNA within an agarose gel in the gel electrophoresis technique is primarily based on what factor?

The size of the DNA fragments

What would the resulting gel look like if your primers did not match a DNA sequence found in your organism or sample?

There would be no bands

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)

These regions are very abundant (many copies/cell) in most organisms sequences flanking these regions are very conserved, so common primers can bind to lots of different taxa

What is the main goal of the "MUSCLE" substop?

This aligns your sequence with other sequences that you select (to look for sequence similarity)

Why do we dye our gels with Gel Red dye?

This dye stains the double stranded DNA in our gel

What is the main goal of the "pair builder" substop?

This tells the computer which reads are the F and R reads from the same samples

Which of following questions could DNA barcoding successfully address?

What species of squid are used in calamari sold in local restaurants?

After running your DNA extract through a nanodrop spectrophotometer you find that your extract has a DNA concentration of 0.1 ng/uL and your A260:280 is 1.80, which of the following should you do to increase the likelihood of your DNA extract PCR amplifying correctly? (If you're torn between two answers, choose the one that requires the least time/effort).

You should do another extraction, your DNA concentration is too low

Phred score

a measure of the quality of the identification of the nucleobases generated by automated DNA sequences

Using AND between two search terms will [ a ] your search Using OR between two search terms will [ b ] your search

a. narrow b. broaden

This is a short summary of a scholarly article that will help you identify whether the article is relevant. This short summary will save you a lot of time, because you won't have to read an entire article before discovering it is not relevant to your topic or interests.

abstract

Your initial DNA extract has a DNA concentration of 425 ng/uL. What volume of this extract would you add to water to get 200 uL of 150 ng/uL DNA solution.

add 70.59 uL of DNA solution to 129.41 uL of water

MUSCLE

an alignment of multiple DNA sequences that allows you to visualize similarities and differences between the DNA sequences

secondary literature

an expert summary or synthesis of someone else's original research that is communicated "second-hand" by people who did NOT collect or analyze the original data

crude alkaline lysis

animal samples fungi samples fish samples insect samples invertebrate samples to amplify meat DNA in dog/cat food

Hook

argument or fact used to justify why anyone should care about the study. used to appeal to the widest audience possible

In the context of our labs, what does "bp" stand for?

base pairs

BLAST

basic local alignment search tool - used to find matching DNA sequences from a database

consensus sequence

combination of trimmed for and reverse DNA sequences that is typically more accurate than each sequence alone

node

common ancestor

The plant DNA extraction protocol includes several extra steps that use isopropanol, ethanol, and TE buffer. What is the overall goal of these "extra" steps?

concentrate and clean your DNA

During this step in PCR, DNA is separated into two single strands.

denaturation

To carry out Sanger sequencing, a mix is needed containing

dideoxyribonucleotides (ddNTPs) DNA polymerase Primer four deoxyribonucleotides (A, T, C, G) DNA template

tertiary literature

encyclopedia entry

What symbol will you see in the sequence viewer if you have inaccurate sequence?

exclamation point in a triangle to the left of your sequence

DNA barcodes are typically useful to determine the genetic variation between individuals of the same species.

false

You cannot balance a centrifuge with an odd number of tubes.

false

You should be sure to put BOTH a forward AND reverse primer in EACH sequencing tube.

false

Why is it important to grind your samples well during DNA extraction?

grinding helps to break open cells and cell membranes to release DNA

PCR reactions that start with very ----- initial concentrations of DNA > 1000 ng/uL are not as likely to work as reactions that start with concentrations of 100 ng/uL

high

aln. length

higher = better match

bit score

higher = better match

branch tips

individual taxa

What are the potential benefits of building a consensus sequence? (check all that apply)

it is more accurate than a single read it can sometimes be longer than a single read (therefore more information)

broad background

larger issue at hand and the most general background

e value

lower = better match

mismatches

lower = better match

μL stands for -----, which is 10^-6 L liters:

microliter

ITS

mushroom

branch length

no meaning

Sheila has noticed that the wasabi (a spicy paste for sushi) she buys from one restaurant is way cheaper than the wasabi she purchases from her favorite restaurant, but the cheaper stuff just doesn't taste as good. After doing some research, she learns that oftentimes horseradish will be dyed green and substituted for real wasabi. She decides to use DNA barcoding to test whether or not the cheap wasabi is horseradish or real wasabi. What kind of study is Sheila doing?

non-experimental

Your initial DNA extract has a DNA concentration of 10 ng/uL. What volume of this extract would you add to water to get 50 uL of 100 ng/uL DNA solution.

not possible

primary literature

original research that is communicated "first-hand" by the people who collected and analyzed the data

red text

outgroup

primary literature

peer-reviewed journal article

RbcL

plant

crude DNA extraction w DNA precipitation

plant samples to amplify plant DNA in dog/cat food

Short, single stranded pieces of DNA that are designed to base pair (or match up with) a specific segment of DNA you want to copy are called:

primers

Original research

research article

Primary article (aka it's about research that the authors did themselves)

research article

Includes LOTS of references

review article

Overview of particular field/topic

review article

Provides good background on a topic

review article

Secondary article (aka it's about research that someone other than the authors did)

review article

Summary of previously conducted research

review article

secondary literature

review article or scientific book

Which of the following are advantages of a review article over a research article? (check all that apply)

review articles provide GOOD SUMMARIES: they summarize many separate studies on a similar topic that have been done by different researchers review articles can help you figure out findings from lots of different studies on your topic

Which of the following is a fundamental difference between a review article vs. a research article?

review articles typically explain work done by other scientists and use this to summarize what's going on in that field or identify larger trends

specific background

specific organism, system, or technique being studied and why they are worth studying

MT-CO1

spider octopus fish mouse cow

For the purposes of this course, what does it typically mean for a sequence to be "high quality" (check all that apply)

the base calls are correct (or at least 99% correct) it is greater than or equal to ~500 bp long

Base call

the nucleotide at a given position in your DNA sequence according to the results of automated sanger sequencing

very specific question/aim/goal

the precise issue that the study is going to tackle

Why are the primers we use to DNA barcode often referred to as "universal" primers?

they work across a wide variety of taxa

Trace file

this depicts the raw results of automated sanger sequencing. Typically include lots of colored humps that represent each nucleotide.

Since dideoxy (Sanger) sequencing is based on chain termination, why are normal dNTPs (deoxyribonucleotides like A, T, G, and C) also included in the reaction?

to produce a range of sizes of DNA synthesis products that terminate at variety of lengths

During the crude alkaline lysis DNA extraction procedure for animal samples, many proteins are precipitated and the DNA remains in solution.

true

Ethanol, proteins, lipids in a DNA extract can potentially inhibit PCR reactions (cause them to fail).

true

Low DNA purity can cause PCR reactions to fail

true

Pipettors are most accurate in the middle to top end of their volume range.

true

You should be sure to include loading dye in each well of your gel that you use, except the well with marker/ladder.

true

accession number

unique identifier # given to each sequence in the BLAST database