Contemporary Lab Skills - Exam 2

What does PCR stand for?

PCR is typically the [blank_start]first[blank_end] step in DNA analysis.

What is the main significance of PCR vs other methods?

PCR amplifies all included DNA segments, which allows us to measure general information about the DNA, such as length and total composition.

What determines the segment of DNA that is amplified by PCR?

DNA amplification after PCR is [blank_start]less[blank_end] than 10^[blank_start]6[blank_end]x

PCR amplification requires [blank_start]little[blank_end] template DNA, but is highly [blank_start]susceptible[blank_end] to contamination.

______________ is a thermostable DNA polymerase used to automate the repetitive steps in the polymerase chain reaction (PCR) technique.

Which of the following is a requirement of PCR?

What kind of primers are used in PCR?

Using dNTP, or [blank_start]deoxynucleotide triphosphate[blank_end], during the [blank_start]extension[blank_end] phase of PCR provides single bases ready to go into DNA and double it, like building blocks.

DNA polymerases (DNAPs)... - Add dNTPs to [blank_start]elongate[blank_end] primer - Require pre-existing [blank_start]oligonucleotide[blank_end] (primer) with free [blank_start]3' -OH[blank_end] - Read DNA template strand & inserts complementary [blank_start]deoxyribonucleotide (dNTP)[blank_end] of the next unpaired nucleotide in the template - Form [blank_start]phosphodiester[blank_end] bond between [blank_start]3' -OH[blank_end] of [blank_start]oligonucleotide[blank_end] & [blank_start]5' -PO4[blank_end] of [blank_start]deoxyribonucleotide (dNTP)[blank_end] - DNA synthesis proceeds [blank_start]5'-->3'[blank_end] - Template strand read [blank_start]3'-->5'[blank_end]

Select all that apply to DNA polymerases for PCR

Match the DNA polymerases with their description [blank_start]Thermus aquatics[blank_end] - Taq Polymerase - In hot springs [blank_start]Thermococcus litoralis[blank_end] - deep ocean vents ...- extends templates >12kb ...- proofreading ability - Manipulation of Vent -->Deep Vent ...- Greater thermostability ...-Lacks exonuclease (exo-) component [blank_start]Thermus maritima (UITma)[blank_end] - Extends long templates - Proofreading ability [blank_start]Thermus thermophilus (Tth)[blank_end] - RT at 70 degrees C + Mn^2+ - similar to RNA-PCR without cDNA [blank_start]Pfu DNA polymerase (marine bacterium)[blank_end] - Proofreading activity - Efficiently incorporates radiolabeled dNTPs - Good for generating DNA probes

How long are typical PCR primers?

Select all that apply to a good PCR primer

Using software such as Oligo and Primer to calculate precise times and temperatures for PCR primer is an option, but calculations made by the researcher should be accurate enough.

Which of the following are major components in a PCR reaction mixture?

Which of the following should be considered when choosing a buffer for PCR?

The PCR cycle has which three steps?

What is the significance of the denaturing phase of PCR?

Denaturation of DNA occurs at what temperature?

Which of the following occurs during the annealing phase of PCR?

Which of the following occurs during the extension phase of PCR

Successive PCR cycles yield fragments precisely delimited by the primer.

Which of the following applies to Agarose Gel electrophoresis (AGE) PCR?

Which of the following applies to Real-time PCR

Label the types of Real-Time PCR

What is the main benefit of using PCR over cloning?

In PCR, "RAPD" stands for... [blank_start]Rapid[blank_end] [blank_start]Amplification[blank_end] of [blank_start]Polymorphic[blank_end] DNA

Which of the following applies to RAPD PCR Analysis?

Which of the following applies to AFLP PCR analysis?

PCR is an effective replacement for cloning

Cloning PCR products involves which of the following?

What are the initial problems associated with Direct PCR sequencing?

What are the solutions for problems associated with direct PCR sequencing?

Select all the denaturants used for direct PCR sequencing

How is one strand selectively removed after synthesis during direct PCR sequencing? (Select 2)

This is a depiction of which kind of PCR sequencing?

This is a depiction of which type of PCR sequencing?

Which of the following applies to PCR cycling?

RT-PCR stands for...

Reverse Transcriptase converts [blank_start]mRNA[blank_end] to [blank_start]cDNA[blank_end] during RT-PCR.

What are the steps of RT-PCR in the correct order? [blank_start]Isolate Poly(A)-mRNA[blank_end] [blank_start]Anneal Poly(dT) Primer to mRNA[blank_end] [blank_start]Reverse Transcriptase Synthesis of cDNA[blank_end] [blank_start]Remove RNA[blank_end]--Use cDNA in PCR

Electrophoresis is...

The migration distance is primarily determined by the ___________ ratio

What is the primary reason we use a matrix in electrophoresis?

A matrix imposes a size constraint on molecule movement during electrophoresis

Select all that are common matrices for electrophoresis

Drag the types of electrophoresis to their applications [blank_start]Starch gel electrophoresis[blank_end] - Proteins Paper electrophoresis - Small molecules [blank_start]Thin-layer electrophoresis (TLE)[blank_end] - Small Molecules [blank_start]Free-flow electrophoresis[blank_end] - Molecules dissolved in liquid [blank_start]Agarose gel electrophoresis (AGE)[blank_end] - DNA (>2 kb) - RNA (>2 kb) [blank_start]Capillary electrophoresis[blank_end] - DNA - RNA -Proteins - Small molecules [blank_start]Polyacrylamide gel electrophoresis[blank_end] (PAGE) - Small DNAs (<2 kb) - Small RNAs (<2 kb)

For nucleic acid gel electrophoresis (separation of DNA and RNA), an [blank_start]agarose[blank_end] matrix is used for general applications while [blank_start]polyacrylamide[blank_end] is used for sequencing and footprinting.

The phosphate in a nucleic acid's backbone has a strong [blank_start]negative[blank_end] charge proportional to the [blank_start]number of base pairs (bp)[blank_end], so it runs towards the [blank_start]positive[blank_end] end of the electrophoresis matrix.

Why is denaturing part of agarose electrophoresis?

Which of the following is used in denaturing agarose gels (DNA)

Which of the following is used in denaturing agarose gels (RNA)

Which of the following is used in denaturing acrylamide gels?

Polyacrylamide gels permit the resolution of individual bases

Pulsed-field electrophoresis (or orthogonal field electrophoresis) does not permit the separation of large DNAs.

Which of the following applies to pulsed-field electrophoresis?

Which of the following are general stains for nucleic acids?

During electrophoresis staining of nucleic acids, the stain intercalates between the ___________ of the nucleic acids.

Alignment of stain molecules to DNA after electrophoresis permits a strong fluorescent signal when stimulated with [blank_start]UV[blank_end].

Proteins are typically separated by what kind of electrophoresis?

PAGE gels can come in what formats?

PAGE buffer systems can either be [blank_start]continuous or discontinuous[blank_end].

Which of the following are components of a polyacrylamide gel?

Which of the following are catalysts used for polymerization of PAGE preparation?

What are the functions of the catalysts used during PAGE preparation?

In PAGE gel preparation the free radicals made from the catalysts form form linkages from [blank_start]acylamide[blank_end] to acrylamide and [blank_start]acrylamide[blank_end] to [blank_start]bis-acrylamide[blank_end].

Which of the following can inhibit APS/TEMED catalysts during PAGE gel preparation?

Select all that apply to %T

Select all that apply to %C

In a discontinuous buffer system... Velocity of Migration = [blank_start]Effective Mobility[blank_end] x [blank_start]Voltage[blank_end] - Proportion of charged molecules determines effective mobility Voltage = [blank_start]Current[blank_end] x [blank_start]Resistance[blank_end] - V = I x R Proteins accumulate in a [blank_start]narrow[blank_end] band (i.e. [blank_start]Stack[blank_end]) before entering the [blank_start]resolving[blank_end] gel Stacking gels have [blank_start]large[blank_end] pores ([blank_start]low[blank_end] %T, [blank_start]no molecular sieving[blank_end]) and a pH of [blank_start]6.8[blank_end]. Glycine is [blank_start]protonated[blank_end], so it has a [blank_start]slow[blank_end] migration and is the [blank_start]trailing[blank_end] ion. Proteins are [blank_start]differentially deprotonated[blank_end], so they have an [blank_start]intermediate[blank_end] migration. Chloride (Cl-) has [blank_start]high[blank_end] mobility and is the [blank_start]leading[blank_end] ion. Cl- moves [blank_start]away from[blank_end] the glycine, creating a [blank_start]low[blank_end] conductivity zone. The [blank_start]low[blank_end] conductivity zone attains a [blank_start]higher[blank_end] voltage gradient. At its steady-state, glycine and Cl- are moving at [blank_start]the same rate[blank_end] with a [blank_start]sharp[blank_end] boundary between them. As the glycine moves through the stacking gel, the [blank_start]Cl-[blank_end]overtakes the [blank_start]proteins[blank_end] [blank_start]in front of[blank_end] the boundary. Proteins have [blank_start]higher[blank_end] mobility than glycine in the trailing [blank_start]high-voltage[blank_end] gradient, so they move [blank_start]at[blank_end] the boundary. Resolving gels have [blank_start]smaller[blank_end] pores ([blank_start]higher[blank_end] %T, [blank_start]molecular sieving[blank_end]) and a pH of [blank_start]8.8[blank_end]. At the resolving gel, glycine [blank_start]deprotonates[blank_end] and [blank_start]increases[blank_end] mobility. The [blank_start]glycine[blank_end] overtakes the [blank_start]proteins[blank_end] and moves just behind the Cl-. Then the Proteins separate based on their [blank_start]charge:mass ratio[blank_end] and molecular sieving due to gel composition (%T, %C).

Denaturing protein electrophoresis is generally conducted using _________ to separate proteins.

The "SDS" in SDS-PAGE stands for...

Select all that apply to the SDS detergent used in SDS-PAGE

SDS-PAGE requires __________ for complete disruption of secondary structure

Which of the following are examples of reducing agents used during SDS-PAGE?

When using an SDS detergent for PAGE... Separation is based on [blank_start]chain length[blank_end] It has a slightly negative charge at pH 7, so the molecules run towards the positive pole

Which of the following applies to Urea gels during denaturing protein electrophoresis?

Which of the following are examples of gel matrices used for non-denaturing protein electrophoresis?

Which of the following are examples of native gels used for non-denaturing protein electrophoresis?

Select all that apply to native gels

Select all that apply to isoelectrofocussing (IEF) gels

Which of the following are examples of protein stains? (Drag to correct spot) Standard: [blank_start]Coomassie Brilliant Blue[blank_end] [blank_start]Ruby Red[blank_end] Most sensitive: [blank_start]Silver[blank_end]

[blank_start]Protein stains[blank_end] (is/are) used for total protein visualization in electrophoresis (steady-state). [blank_start]Autoradiography[blank_end] (is/are) used for visualizing newly synthesized proteins after electrophoresis.

Select all that apply to Autoradiography

Rf = Distance of [blank_start]Migrated Band[blank_end] / Distance of [blank_start]Migrated Dye[blank_end]

Migration in SDS-PAGE should be determined with a _________ function

Rf is typically used when molecular weight markers (MWM's) run on the gel with the sample.

2D enhanses esolution over 1D PAGE.

Why do we use 2-Dimensional Gel Electrophoresis?

Select all that apply to protein separation in the first dimension (electrophoresis).

Select all that apply to protein separation in the second dimension (electrophoresis).

Why do posttranslational modifications occur after separation in the second dimension (electrophoresis)>

Select all components of In vitro translation systems (electrophoresis)

What are the uses of In vitro translation of proteins?

What is the basic procedure of In vitro translation of proteins? 1. [blank_start]Extract mRNAs[blank_end] 2. [blank_start]Translate mRNAs In vitro[blank_end]

___________ is the transfer of macromolecules from gel to membrane filter following electrophoresis.

What are some of the advantages of analyzing macromolecules using probes instead of in gels?