BIOL 222 Lecture 8: PCR and DNA Sequencing

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21 Sep 2016

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Pcr = polymerase chain reaction: developed in 1985 by kary mullis, method you can amplify (copy) a speci c region of dna. Ingredients for pcr: template dna (of what you want to copy), polymerase, dntps (all four nucleotides), and primers. Dntp = dttp, datp, dctp, and dgtp. Two kinds of primers are used forward primer and a reverse primer: bind to opposite sides of the template strands, functions of these ingredients. The two primers provide a free 3" end for polymerase to bind to: these primers also have a speci city on where they bind, which allows us to control what the polymerase will copy. Steps of pcr: 1. heat the template strands to 95 c to denature and separate the two strands by breaking the hydrogen bonds. In humans this role is done by helicase: 2. primer annealing. 1min/thousand bp ~72 : 4. repeat (denature, anneal, extend)

Related Questions

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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

Answered most of the questions. Can you tell me if my answers are correct? Last question of the month. Thank you. I appreciate it.

4. You perform electrophoresis on the product of a PCR and see that instead of the 300-base band you expected that there are five different bands.

What controls the specific sequence to which a PCR primer binds? Complementary sequence and annealing temperature control the specific sequence to which a PCR primer binds.

What are two factors that contribute to the annealing temperature of the primer? Two factors that contribute to the annealing temperature of the primer is primer length and the Guanine and Cytosine content.

What part of your thermal cycle protocol could you change, and how would you change it, to try and get rid of the extra bands?

To get rid of the extra band, we have to increase the annealing temperature.

If, after performing electrophoresis, there were no bands on the gel, what part of your thermal cycle protocol would you change, and how would you change it?

After performing electrophoresis, if there were no bands on the gel then we have to decrease the annealing temperature of the primer

5. If you are using a DNA sequence from a related species to design primers for your species of interest, why is it better to design your primers to complement an exon?

In related species there are similarities but their genetic makeup will not be the same. It is better to design your primers to complement an exon because ??

6. Why is it better to use a 25-base primer than a 10-base primer? Why do you have to have a lower annealing temperature for a 10-base primer?

It is better to use a 25-base primer than a 10-base primer because a 10-base primer is not long enough and may pair with a wrong portion of DNA, which would give wrong results. You have a lower annealing temperature for a 10-base primer because annealing temperature increases when the number of base pairs increase. If the temperature is too high the primer might not bind.

Identify the three steps in PCR and tell the function of each step.

Denaturation - when the double-stranded DNA is heated to 92-95â for about one minute, to separate it into two single strands.

Annealing - temperature of the reaction is lowered to a temperature that is between 45â and 65â to allow the DNA primers to attach to the template DNA.

Extension - temperature of the reaction is raised to a temperature between 45â and 65â and the new strand of DNA is made by adding nucleotides to the ends of the primers.

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 222 Lecture 8: PCR and DNA Sequencing

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