Bio 219 Final Exam Review

After PCR: Tube contents

-Synthesized DNA

-Excess dNTPs

-Excess primers

Shotgun sequencing

Breaking apart large strands of DNA and sequencing the smaller pieces individually and assembling them via their overlapping ends

Bridge PCR

Amplification of DNA is done in conjunction with primers that are attached to a solid surface to make DNA colonies that can be examined later

What do double peaks indicate on a chromatogram?

usually indicate SNPs or single nucleotide polymorphisms. This suggests that the sequence you have can exist in multiple forms. This may be due to base changes that do not result in an altered amino acid or negligible changes such as one non-polar amino acid for another non-polar amino acid.

You notice on the PCR Gel that in the lane with pGEM, without an insert, you see a bright, small MW band near the bottom of the lane (< 100 bp). What could this be?

a. RNA contamination

b. gadA gene product

c. Supercoiled vector

d. Primer Dimers

e. luxI gene product

d.

Primer Dimers

What does the "Exo" in the ExoSAP step in PCR clean up do?

a.

Shortens PCR products so they are easier to determine

b.

Removes chDNA leftover from the plasmid prep

c.

Removes excess Primers

d.

Elongates any unsynthesized strands

e.

Removes the excess dNTPs and the Polymerase

c.

Removes excess Primers

In the Sanger Sequencing method what is crucial for nucleotide detection?

a.

DNA with low GC content.

b.

dNTPs conjugated to a fluorophore

c.

Primers conjugated with a fluorophore

d.

ddNTPs conjugated to a fluorophore

e.

PCR products longer than 500bp

d.

ddNTPs conjugated to a fluorophore

When analyzing a chromatogram what are the two main aspects to look out for?

a.

Miscalled Nucleotides

b.

Stretches of the same nucleotide

c.

Number of Nucleotides Present

d.

Background Noise

e.

BLAST anchors

a.

Miscalled Nucleotides

d.

Background Noise

True or False. You can sequence a PCR Product that has not been cleaned up, but it is likely to have significant background "noise" that leads to more errors and miscalled bases.

True

Double peaks on a chromatogram usually indicate

a.

DMPs

b.

ARPs

c.

SNPs

d.

ABCs

e.

FMPs

c.

SNPs

In a situation where there seems to be a space between nucleotide peaks, it common practice to indicate that space with a "P" to indicate it is an unknwon purine or pyrimidine.

False

In the ExoSAP clean-up, indicate what "Exo" and 'SAP" are in the product.

E.Coli Exonuclease I

Shrimp Alkaline Phosphatase

Which of the following statements is NOT true about ddNTPs?

a.

It terminates the DNA sequence.

b.

ddNTPs are nucleotides used in the Sanger Sequencing method.

c.

It is used in Sanger Sequencing method as a chain terminating nucleotide.

d.

ddNTPS cannot form a phosphodiester bond.

e.

Contains a 3' OH group in the deoxyribose sugar

e.

Contains a 3' OH group in the deoxyribose sugar

In the Sanger sequencing method, there should be a higher concentration ddNTP's than dNTP's to ensure that termination occurs at every occurrence of that particular nucleotide.

False

The positive control pGEM lux+ plamsids are pGEM 500 (forward insertion of the lux operon) and pGEM 501 (reverse insertion of the lux operon). In PCR amplification of the luxI gene from the pGEM 500 we only need to use forward primers because the operon inserted in the forward direction.

False

What is the purpose of the ExoSAP clean-up following a PCR reaction?

-To remove the excess dNTPs and primers or else they will cause background noise

Exo- E.coli Exonuclease I

-Catalyzes the removal of ssDNA from the 3'→5' end direction = removes primers

SAP- Shrimp Alkaline Phosphate (SAP) - Binds up free nucleotides (dNTPs)

What is Sanger sequencing?

Another PCR of you results with the first PCR product as a template

-The PCR is normal except for the addition of ddNTPs = no 3' OH so no elongation

-The DNA will get sequenced and randomly a ddNTP will get added, stopping that strand

-smallest fragment = the first nucleotide of the sequence

What is a chromatogram?

The fluorophore on each ddNTP will be detected as it passes through the filter and recorded until an entire sequence read is detected. This will produce the nucleotide sequence of your results

What does an "N" designate on a chromatogram result?

A base-caller may attempt to "name" that space and sometimes may put a N there

What does BLAST stand for and for what is it used?

Basic Local Alignment Search Tool

-Find regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches.

What is the most common method to extract RNA

The most common method is acid guanidinium thiocyanate-phenol-chloroform extraction(high purity and high recover of RNA)

What do we know about our cultures we are extracting RNA from?

1. Culture A: E. Coli + pGEM lux-

pGEM does not have the lux operon so no AHL is made, therefore E.Coli should not display any changes in gene expression of GadA

2. Culture B: E.coli + pGEMlux+

pGEM does have the lux operon so AHL is made, therefore we should see an up regulation of GadA, our target gene

True or False. Due to the presence of the luxR gene, gene expression of the target gene, gadA, should decrease in the lux+ clones.

False

Isolating RNA instead of DNA will provide what?

a.

A snapshot of the cDNA in the cell

b.

A snapshot of the mutations in the genome

c.

A snapshot of the proteins available at that time

d.

A snapshot of the genes being transcribed at that time

d.

A snapshot of the genes being transcribed at that time

What feature of RNA do RT primers take advantage of?

a.

5' Methyl guanosine cap

b.

3' Poly-A tail

c.

Uracil instead of thymine in RNA

d.

RNA Polymerase

e.

Secondary RNA structures

b.

3' Poly-A tail

True or False. During cDNA synthesis the RNA template is degraded.

True

Which polymerase is involved during cDNA synthesis?

a.

qRNA polymerase

b.

cDNA Polymerase

c.

DNA Polymerase

d.

RNA Polymerase

e.

Cyclical Polymerase

c.

DNA Polymerase

In which culture(s) will AHL (N-acyl homoserine lactone) be made?

a.

E. coli + pGEMlux-

b.

E. coli + pGEMlux+

c.

Both E. coli + pGEMlux- and E. coli pGEMlux+ cultures

b.

E. coli + pGEMlux+

______________________ is used to synthesize a single strand of DNA complementary to the original mRNA strand for RT-PCR.

Reverse Transcriptase

Which gene is the luxR homolog in E. Coli?

a.

RckL

b.

SdiA

c.

SrgD

d.

SrgA

e.

GadA

b.

SdiA

What is used as template for making the 2nd strand of the cDNA in RT-PCR?

a.

the newly synthesized DNA strand

b.

the original mRNA is used

c.

another random strand of DNA is added

d.

RT-PCR can only generate ssDNA products

a.

the newly synthesized DNA strand

End-point (regular) PCR and RT-PCR differ in that, End-point PCR requires the use of dNTPs whereas RT-PCR requires the use of ddNTPs.

False

The Wash Buffer in the RNeasy prep contains ______________ to remove salts and organic molecules.

a.

Chaotropic Salt

b.

EDTA

c.

Lysozyme

d. β-Mercaptoethanol

e.

Ethanol

e.

Ethanol

In a traditional Phenol Extraction a low ~pH 8 favors the separation of DNA and RNA leaving the RNA in the aqueous phase and DNA precipitated at the interface between the aqueous and organic phases.

False

RNAprotect Reagent is needed when trying to isolate RNA to enable RNase inactivation, stabilization of the RNA and preservation of the RNA expression pattern because RNA is less stable than DNA.

True

Why did we extract RNA rather than DNA for analyzing GadA gene?

Because all actively transcribed genes of DNA will be turned into RNA, then protein. Therefore, if we collect RNA we can see what is being transcribed at that time. Basically this is going to be collection of genes being used.

Once RNA was extracted, we converted our product into cDNA using RT-PCR. This process includes (4) distinct steps, as outlined in lecture. What are these steps?

1. using the poly-A-tail a primer with a complementary sequence is attached

2. Reverse Transcriptase is used to synthesize a single strand of DNA complementary to the original mRNA strand.

3. The RNA template is then degraded

4. The newly synthesized DNA strand folds back on itself and acts as it own primer, allowing the DNA polymerase to synthesize a double stranded piece of DNA

What kind of primer was used in converting RNA to cDNA?

Oligo(dT) primer

During the RNeasy miniprep, we utilized lysis buffer that contained six important reagents. What are they, and why were they necessary?

1.Tris buffer

-Maintains pH; contains EDTA, which chelates divalent cations (Mg⁺ and Ca²⁺) to prevent DNase from degrading plasmid

2. Lysozyme

-Breaks down the cell walls of bacteria by destabilizing peptidoglycan

3. Detergent

-SDS, Triton X: lyses the cell membrane by destabilizing the lipopolysaccharide layer

4. Proteinase K

-Proteinase K is an enzyme that will aid in the release of nucleic acids while deactivating nucleases (enzymes that degrade nucleic acids) that are present. The addition of proteinase K degrades these nucleases and protects the nucleic acids from nuclease attack

5. Beta-Mercaptoethanol

-Degrades ribonuclease by breaking disulfide bonds making the proteins unfold and become non-functional

6. Chaotropic Salts (Guanidium Isothiocyanate)

-Maintains a hydrophobic solution by preventing hydrogen bonding between RNA and water and setting up conditions to bind to the membrane

During the RNeasy miniprep- Wash and Elution

1.Wash Buffer + 90% EtOH-Buffers RW1 and RPE-Salts and small organic molecules are soluble in ethanol so they go into solution while RNA stay bound to the silica-Contaminants are then removed by centrifugation2. Elution Buffer-RNAse-Free Water-Rehydrates RNA and frees it from the silica membrane

In a Real Time PCR application...

(select all that apply)

a.

you are relating the end value to how much mRNA was originally made.

b.

you start with DNA.

c.

you are aiming to get a concentrated amount of DNA.

d.

you are analyzing product in real time.

a.

you are relating the end value to how much mRNA was originally made.

d.

you are analyzing product in real time.

Real Time PCR can be:

a.

both qualitative or quantitative

b.

long and tedious

c.

qualitative only

d.

quantitative only

a.

both qualitative or quantitative

True or False. qPCR dyes, such as SYBR Green, fluoresce when bound to ssDNA.

False

Looking at an amplification curve generated from qPCR, what is the term used to describe the cycle at which fluorescence of a dye is detectable?

a.

Cq Value

b.

Cycle Value

c.

Threshold

d.

Exponential Phase

a.

Cq Value

Four bacterial cells were placed in separate cultures. To each culture a different concentration of AHL was added. When in the presence of an AHL each bacterial cell will induce transcript of Gene X. After collecting RNA from each culture, 30 minutes post-exposure to their specific AHL concentration, a RT-PCR and qPCR was carried out. When looking at the amplification curve generated by the qPCR for Gene X, four lines are noticeable; a Red, a Blue, a Violet, and a Green. Looking at the Cq values for each results in the following:

Red Line = 24.6

Blue Line = 10.3

Violet Line = 32.3

Green Line = 93.7

Which of the four lines represents the culture that had the most concentrated AHL added to their medium?

Blue

For the qPCR that we are performing this week in lab, we are measuring the increase in quantity of the [x] transcript in the presence of the [y] inducer from the [z] operon.

Specified Answer for: x

GadA

Specified Answer for: y

AHL

Specified Answer for: z

lux

In two step reactions where the RT-PCR and the qPCR are performed separately, what type of primers are used in qPCR?

a.

Random sequence primers

b.

Poly A primers

c.

Oligo(dT) primers

d.

Sequence specific primers

d.

Sequence specific primers

For the qPCR we are performing in lab this week, which culture should have the lowest Cq value?

a.

E. coli + pGEMlux-

b.

E. coli + pGEMlux+

b.

E. coli + pGEMlux+

What primers can be used for the Reverse Transcriptase PCR? Select all that apply.

Oligo ddA primers

Oligo dA primers

Oligo ddT Primers

Oligo dT Primers

Random primers

Gene specific primers

Oligo dT Primers

Random primers

Gene specific primers

A 1.8 fold increase would represent an increase of [x]%.

180

To determine the Ratio of Expression, you first need to calculate the Relative Expression which is determined by the equation:

2(Cq(Target)-Cq(Ref))

False

A high Cq value means there was more cDNA of that region of DNA to start with, which posits there was more mRNA made.

False

What are the purposes of both conventional PCR and real time PCR? (hint: the readouts they produce are used differently)

Conventional PCR

-Starts with DNA

-Amplicon is detected and analyzed at an end-point analysis

-Analyzing finished product

-Usually get a concentrated amount of DNA

Real Time PCR

-Start with cDNA

-Amplicon is based off of already existing concentration of nucleic acid

-Analyzing product, in real time

-Usually use to look at how much transcription was made

qPCR utilizes fluorescent dyes to quantify DNA. Which dye did we use?

SYBR Green

What is the significance of the Cq value generated by qPCR?

The Cq values is the point at which fluorescence accumulates enough to be detected

You completed qPCR and generated a curve for three samples. Your resulting Cq values are 15.34, 30.02, and 12.14. Which of these had the highest concentration of RNA template? How do you know?

12.14 would have the highest concentration of RNA since it has a low, or early, Cq value meaning that not many cycles were needed for fluorescence detection