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2. This question concerns PCR and DNA sequencing. a. Describe what would happen in a PCR experiment if you only used one primer instead of two primers. b. Suppose you perform PCR using only one copy of a double-stranded DNA molecule. How many copies of this double-stranded DNA molecule would be present after 15 cycles? c. We usually think of enzymes being the most active at around 37?C (near body temperature). In PCR, however, DNA synthesis occurs at 72?C during each cycle. What is unique about the DNA polymerase that is used in PCR? d. Dideoxynucleotides (ddNTPs) play an important role in DNA sequencing. What is the structural difference between a nucleotide (NTP), a deoxynucleotide (dNTP), and a dideoxynucleotide (ddNTP).

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Collen Von
Collen VonLv2
28 Sep 2019

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Related questions

The PCR (polymerase chain reaction) protocol that is currently used in laboratories was facilitated by the discovery of a bacterium called Thermus aquaticus in a hot spring inside Yellowstone National Park, in Wyoming. This organism contains a heat-stable form of DNA polymerase known as Taq polymerase, which continues to function even after it has been heated to 95°C. Why would such a heat-stable polymerase be beneficial in PCR?

What would happen if it weren’t heat stable?

How might you choose a region of DNA for a PCR primer so as to increase the temperature necessary for primer annealing (to minimize nonspecific PCR products)?

A PCR reaction begins with 5 double stranded segment of DNA. Estimate the number of double-stranded copies of DNA that are present after the completion of 15 amplification cycles?

Please answer all

Why does a template-dependent DNA polymerase require a primer to initiate DNA synthesis?

DNA polymerase require 5' phosphate to add a new nucleotide

DNA polymerase require 3' hydroxyl group to add a new nucleotide

DNA polymerase require energy by hydrolizing the primers

Which of the following enzymes is template independent?

Reverse transcriptase

Klenow fragment

DNA polymerase

Terminal deoxynucleotidyl transferase

RNA polymerase

The natural function of a DNA ligase enzyme is:

Joining together DNA sequences with compatible cohesive ends from two different organisms to yield a recombinant DNA molecule

Sealing single stranded nicks in double stranded DNA molecules

Joining together two blunt ended fragments of DNA within a cell

Joining together two single stranded DNA molecules

The natural function of 3' -> 5' exonuclease activity of a DNA polymerase is to:

Remove the 5' end of the DNA strand that is being copied

Remove damaged nucleotide from the template strand

Remove nucleotides from the end of DNA to generate blunt ends

Remove incorrect nucleotides from the newly synthesized DNA

Which technique is used to resolve the different sizes of DNA fragments?

Gene cloning

PCR

DNA sequencing

Gel electrophoresis

Which of the following vectors does not contain bacterial origin of replication (oriC)?

plasmid

cosmid

bacterial artificial chromosome

lambda vector

Which process of bacteriophage is not used for gene cloning?

Concatemer formation

Lytic cycle

Lysogenic cycle

viral packaging

What vector would be best suited for creating a contig of bovine (cattle) chromosome 10?

lambda vector

Cosmid vector

P1 vector

Plasmid

E. coli takes up plasmid DNA by which of the following methods?

Transduction

Transformation

Conjugation

Transfection

The following times and temperatures are an example of the steps for PCR. You can use the Figure to help you answer the following questions.

Why is the first step is carried out at 94°C?

To degrade the template

To denature the template

To activate the polymerase

To facilitate the primer binding

What happens in the reaction when the temperature shifts to 55°C during cycling?

the DNA polymerase will carry out DNA synthesis by extending the annealed primers

the DNA polymerase will finish DNA synthesis

the primers anneal to the single-stranded regions of the DNA

the primers anneal to the double-stranded regions of the DNA

During cycling, what occurs when the temperature is at 72°C?

the primers anneal to the double-stranded regions of the DNA

the DNA polymerase will carry out DNA synthesis by extending the annealed primers

the primers anneal to the single-stranded regions of the DNA

the DNA polymerase will finish DNA synthesis

You designed a set of primers to amplify 1 kb DNA with polymerase chain reaction. By which cycle of polymerase chain reaction, we would expect to see the first double stranded DNA with expected size?

The end of cycle 1

The end of cycle 2

The end of cycle3

The end of cycle 4

Evaluation of PCR product electrophoresed on a 0.8% agarose gelshows non-specific bands. The appropriate modification for the nextPCR reaction is to


a )increase the concentration of Taq polymerase.

b) increase the number of PCR cycles.

c) decrease the template denaturation temperature.

d) reduce the concentration of primers.


In the Sanger sequencing method, chain termination is the resultof


a)misincorporation of dUTP instead of dTTP.

b)incorporation of ddGTP.

c)secondary structures in the template strand.

d)sequencing through repeats.


The purpose of EDTA in a DNA storage buffer is to


a)maintain single strands.

b)bind magnesium

c)activate DNases.

d)dissolve DNA into solution.


The thermal cycler ramp refers to the


a)degree of incline of the machine on the laboratory bench.

b)gradient between denaturation and annealing temperature.

c)programming key to set number of PCR cycles.

d)amount of time required to reach the desired temperature.


Evaluation of a blood sample indicates the presence of a clot.Corrective action to salvage this specimen for analysis would mostlikely require


a)digestion with proteinase K.

b)homogenizing the sample under UV irradiation.

c)removal of the clot with urea.

d)requesting a new sample immediately.


Incorporation of biotin into template DNA by nick-translationrequires


a)DNase.

b)ligase.

c)random hexanucleotides.

d)3 temperatures.


An isolated DNA sample is viscous, difficult to pipet, and willnot resuspend in TE buffer after rehydration at 37C for 24 hours.The most appropriate step is to


a)digest the DNA with a frequent cutting restriction enzyme.

b)re-extract the original sample with higher volumes ofreagents.

c)increase the rehydration temperature to 95C.

d)add more TE buffer.


Excess glycerol in a restriction digestion reaction resultsin


a)enzyme degradation.

b)diminished enzyme activity.

c)increased temperature required for digestion.

d)decreased enzyme buffer pH.


Primer dimers will most likely be a result of PCR when


a)3' and 5' primers are designed as 50% GC.

b)DNA template concentration is very low.

c)enhancing agents are added to the master mix.

d)template DNA is initially denatured completely.


Correct operation of a pH meter requires


a)calculation of the electrode millivolt equivalents.

b)standardization of the meter with a range of buffers.

c)documentation of absorbance and standard deviation.

d)verification of pH readings with calf thymus DNA.


The uracil DNA glycosylase pre-PCR decontamination procedurefailed. The most likely cause of the failure is the


a)enzyme solution was heated prior to PCR.

b)enzyme buffer solution was alkaline pH.

c)N-glycosidic DNA bonds cleaved after enzyme treatment.

d)PCR was performed with dTTP instead of dUTP.


The real-time PCR analysis does not show the characteristicmelting curve for the patient or control samples. The fluorimetryreadings of the PCR products indicate the appropriate concentrationof template was added to the real-time PCR reaction mix. Which ofthe following reagents was likely missing from the PCR master mixto account for this finding?


a)SYBR Green I

b)template DNA

c)DMSO

d)5X Cresol Red


Which of the following conditions favor enhanced specificity ofPCR?


a)decrease in the concentration of dNTPs

b)increase in the sodium chloride concentration

c)increase in the concentration of Taq polymerase

d)decrease in the ramp speed and temperature


Which of the following compounds is an inhibitor of Taqpolymerase?


a)magnesium

b)hemoglobin

c)DMSO

d)betaine


Which of the following compositions of gels would allow for theefficient separation of linear DNA molecules in the range of 100 to300 base pairs in length?


a)0.3% agarose

b)1.0% agarose

c)1.0% polyacrylamide

d)8.0% polyacrylamide


4.92*10^4 cpm of a random primed 32P dCTP (3000 Ci/mmol) labeledoligonucleotide probe were precipitated from a 50 uL standardreaction by TCA and 5.28*10^4 cpm is the total cpm in the sample.What is the percent incorporation of radioactivity for thislabeling reaction?


a)1.86%

b)46.50%

c)93.00%

d)98.40%


The stock solution of HCl is 1M. How much stock solution isrequired to prepare 250 mL of 50 mM HCl?


a)0.2 mL

b)5 mL

c)12.5 mL

d)125 mL


Spectroscopy measurement of a DNA sample isolated by saltextraction technique gave the following absorbance readings: A2600.4032 A280 0.3765

This sample is:


a)acceptable for molecular testing.

b)contaminated with salt.

c)contaminated with protein.

d)contaminated with high amounts of RNA.


What is the dilution factor if 50 uL of blood is diluted with950 uL of saline?


a)19

b)20

c)40

d)50


Which of the following are used to prepare a 0.9% agarosegel?


a)0.9 g agarose + 100 mL distilled water

b)0.9 g agarose + 100mL TAE

c)9 g agarose + 100mL TBE

d)90 g agarose + 100 mL buffer


The concentration of double-stranded DNA with an optical densityreading of 1.0 is


a)0.5 ug/ul

b)1.0 ug/ul

c)10 ng/ul

d)50 ng/ul


How much Tris base (MW 121.1) would be required to make 4 litersof Tris-EDTA buffer that is 15 mM Tris?


a)0.7266 grams

b)0.1211 grams

c)1.8165 grams

d)7.266 grams

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