BIOL1130 Lecture Notes - Lecture 8: Polymerase Chain Reaction, Dna Replication, Deoxycytidine Triphosphate

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orangegoat71

7 Jun 2018

School

UWA

Department

Biochemistry and Molecular Biology/Biology

Course

BIOL1130

Professor

Guijun Yan

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Module 3 Assessments: DNA review

DNA replication

• Absolute requirements for DNA replication:

1. A pre-existing DNA template

2. A pre-existing free 3 hydroxyl group on a primer

3. A protein catalyst (an enzyme)

a. DNA polymerase

4. dNTP precursors (building blocks)

a. dNTP= dATP, dCTP, dGTP, dTTP

• this reaction can occur in a test tube if the temperature, pH and salt

conditions meet the needs of the enzyme.

• Hence, DNA sequencing and Polymerase Chain Reaction (PCR)

DNA sequencing

• DNA sequencing is interrupted replication

• The sequence obtained is the reverse and complement of the single

stranded template provided and extends from the hybridized primer

• DNA polymerase is given a mix of normal dNTPs (dioxy) and colour

labeled ddNTPs (di-dioxy that lack the 3 –OH (causing termination)

• A random mix of replication products is obtained, of all possible lengths,

with colour coded by the termination ddNTP

• These are separated by size (length) to read the sequence

DNA Replication: PCR (Polymerase Chain Reaction)

• The generation of short fragments of DNA from a double stranded

template is done using the method of polymerase chain reaction (PCR)

• Genomic DNA the template is heated to 95˚C to denature the template

(break the hydrogen bonds)

• The mix is cooled enough so that very short primers can hybridise to their

complementary sequences but the larger DNA fragments dont rejoin

• DNA polymerase can then replicate each strand

• The reaction is then repeated; now the copies are also copied but only

between the primer sequences

• After about 30 repeats, the short replicated fragment is present in very

large numbers and can be cloned/directly sequenced.

Identification through DNA

• Due to amplification steps, a very small amount – even one molecule of

DNA is enough to identify someone

• Primers are used that amplify a number of DNA fragments

• DNA fragments are separated by gel electrophoresis

• Single nucleotide mutations in DNA sequence between individuals

generates diversity in the banding pattern

o Either the primer sequence has changed so a band does not form

o Or the length of DNA between the primers that bracket the band

has changed

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Related Questions

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

jadeturtle730

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

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BIOL1130 Lecture Notes - Lecture 8: Polymerase Chain Reaction, Dna Replication, Deoxycytidine Triphosphate

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