BIOL 2500 Lecture Notes - Lecture 20: Thermus Aquaticus, Dna Replication, Dideoxynucleotide

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pucealligator102

28 May 2016

School

University of Manitoba

Department

Biological Sciences

Course

BIOL 2500

Professor

Roman Gietz

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Document Summary

Molecular genetic analysis methods make use of dna replication processes. Molecular biologists have used their understanding of dna replication to develop new methods of molecular analysis. Two widely used methods include polymerase chain reaction (pcr) and dideoxynucleotide. The polymerase chain reaction (pcr) is an automated version of dna replication that produces millions of copied of a short target dna segment. Pcr reactions are carries out in small volumes. A double-standee dna template containing the large sequence to be ampli ed. A supply of the four dna nucleotides. The most commonly used dna polymerase, taq is isolated from thermus aquaticus, which occurs naturally in hot springs. Pcr uses tow dna sequences called pcr primers that proved a starting point for taq polymerase to add nucleotides. The pcr primers de ne the 5" and 3" boundaries of the replication products. Pcr is composed of three steps that result in exponential ampli cation of large numbers of the target dna.

Related Questions

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 94Â°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 55Â°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 72Â°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 gel shows non-specific bands. The appropriate modification for the next PCR reaction is toa )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 result ofa)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 toa)maintain single strands.b)bind magnesiumc)activate DNases.d)dissolve DNA into solution.The thermal cycler ramp refers to thea)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 most likely requirea)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-translation requiresa)DNase.b)ligase.c)random hexanucleotides.d)3 temperatures.An isolated DNA sample is viscous, difficult to pipet, and will not resuspend in TE buffer after rehydration at 37C for 24 hours. The most appropriate step is toa)digest the DNA with a frequent cutting restriction enzyme.b)re-extract the original sample with higher volumes of reagents.c)increase the rehydration temperature to 95C.d)add more TE buffer.Excess glycerol in a restriction digestion reaction results ina)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 whena)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 requiresa)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 procedure failed. The most likely cause of the failure is thea)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 characteristic melting curve for the patient or control samples. The fluorimetry readings of the PCR products indicate the appropriate concentration of template was added to the real-time PCR reaction mix. Which of the following reagents was likely missing from the PCR master mix to account for this finding?a)SYBR Green Ib)template DNAc)DMSOd)5X Cresol RedWhich of the following conditions favor enhanced specificity of PCR?a)decrease in the concentration of dNTPsb)increase in the sodium chloride concentrationc)increase in the concentration of Taq polymerased)decrease in the ramp speed and temperatureWhich of the following compounds is an inhibitor of Taq polymerase?a)magnesiumb)hemoglobinc)DMSOd)betaineWhich of the following compositions of gels would allow for the efficient separation of linear DNA molecules in the range of 100 to 300 base pairs in length?a)0.3% agaroseb)1.0% agarosec)1.0% polyacrylamided)8.0% polyacrylamide4.92*10^4 cpm of a random primed 32P dCTP (3000 Ci/mmol) labeled oligonucleotide probe were precipitated from a 50 uL standard reaction by TCA and 5.28*10^4 cpm is the total cpm in the sample. What is the percent incorporation of radioactivity for this labeling reaction?a)1.86%b)46.50%c)93.00%d)98.40%The stock solution of HCl is 1M. How much stock solution is required to prepare 250 mL of 50 mM HCl?a)0.2 mLb)5 mLc)12.5 mLd)125 mLSpectroscopy measurement of a DNA sample isolated by salt extraction technique gave the following absorbance readings: A260 0.4032 A280 0.3765This 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 with 950 uL of saline?a)19b)20c)40d)50Which of the following are used to prepare a 0.9% agarose gel?a)0.9 g agarose + 100 mL distilled waterb)0.9 g agarose + 100mL TAEc)9 g agarose + 100mL TBEd)90 g agarose + 100 mL bufferThe concentration of double-stranded DNA with an optical density reading of 1.0 isa)0.5 ug/ulb)1.0 ug/ulc)10 ng/uld)50 ng/ulHow much Tris base (MW 121.1) would be required to make 4 liters of Tris-EDTA buffer that is 15 mM Tris?a)0.7266 gramsb)0.1211 gramsc)1.8165 gramsd)7.266 grams

crimsoncaribou758

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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BIOL 2500 Lecture Notes - Lecture 20: Thermus Aquaticus, Dna Replication, Dideoxynucleotide

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