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6. (10 points) When double-stranded DNA is heated, the two strands separate into single strands in a process called melting or denaturation. The temperature at which half of the duplex DNA molecules are intact and half have melted is defined as the Tm.

a. Do you think Tm is a constant, or can it depend on other small molecules in the solution? Do you think high salt concentrations increase, decrease, or have no effect on Tm

b. Under standard conditions, the expected melting temperature in degrees Celsius can be calculated from the equation Tm = 59.9 + [0.41 × %(G + C)] – (675/length of duplex). Does the Tm increase or decrease if there are more G + C (and thus fewer A + T) base pairs? Does the Tm increase or decrease as the length of DNA increases? Why?

c. Calculate the predicted Tm for a stretch of double helix that is 100 nucleotides long and contains 50% G + C content.

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11 Dec 2018

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Related questions

Which of the following did Erwin Chargaff observe?

A)

Base composition changes as the organism ages.

B)

Base composition of DNA does not vary from one species toanother.

C)

In all species the number of adenines equals the number ofguanines.

D)

In all species A + T = G + C.

E)

DNAs isolated from different tissues of the same species havethe same base composition.

The melting temperature (Tm) of a DNA duplex is:

A)

the temperature at which double-stranded nucleic acids degradeinto free nucleotides.

B)

the temperature at which double-stranded DNA is toosingle-stranded to ever renature back to double strands.

C)

the temperature at which double-stranded DNA is completelymelted.

D)

the temperature at which double-stranded DNA has become 50%single-stranded.

E)

the temperature at which double-stranded DNA begins to melt.

Given your knowledge and the descriptions of each of thesemethods, which of the following does NOT rely on the ability ofnucleic acids to hybridize with each other?

A)

Southern blotting for the detection of specific DNA fragmentsthat have been separated by gel electrophoresis.

B)

Colony blot hybridization to identify a specific sequence from acollection of sequences that have been inserted into bacteria.

C)

Northern blotting for the detection of specific RNA fragmentsthat have been separated by gel electrophoresis.

D)

A DNA oligonucleotide microarray incubated with probes made fromcDNA.

E)

Western blotting for the detection of specific proteins thathave been separated by gel electrophoresis.

Which of the following is a possible reason why DNA uses thymineinstead of uracil?

A)

Cytidine is deaminated to uracil so it would be difficult forany repair mechanism to differentiate between uracil that belongsin the sequence and uracil that resulted from deamination.

B)

Adenine is deaminated to uracil so it would be difficult for anyrepair mechanism to differentiate between uracil that belongs inthe sequence and uracil that resulted from deamination.

C)

When uracil is bound to a deoxyribose it will become morereactive, forming covalent bonds with other bases.

D)

None of the above.

Which of the following is an enzyme used in cloning to breakcovalent bonds?

A)

DNA ligase

B)

Restriction endonuclease

C)

Reverse transcriptase

D)

DNA polymerase I

E)

Alkaline phosphatase

Which of the following could you use to remove the 5' phosphatesafter cleavage of a plasmid with a single type of restrictionenzyme to ensure that the plasmid would not simply fuse backtogether upon addition of ligase?

A)

Reverse transcriptase

B)

Alkaline phosphatase

C)

DNA ligase

D)

DNA polymerase I

E)

Restriction endonuclease

Which of the following would you use to create a cDNA librarythat you would not use for a genomic library?

A)

Alkaline phosphatase

B)

Reverse transcriptase

C)

Restriction endonuclease

D)

DNA ligase

E)

DNA polymerase I

A plasmid vector typically has which of the followingfeatures?

A)

An origin of replication

B)

A selectable marker to ensure that the only bacteria growingcontain the intact or recombinant vector.

C)

Unique restriction sites for insertion of DNA.

D)

Small size to facilitate entry into the cell and biochemicalmanipulation of the DNA.

E)

All of the above are features of plasmid vectors.

Which of the following is FALSE of genomic or cDNAlibraries?

A)

Genes represented in cDNA libraries include control sequencesthat direct transcription.

B)

cDNA libraries contain cDNAs made from mRNA.

C)

Genomic libraries contain fragments of genomic DNA that aregenerated by partial digests with restriction enzymes.

D)

Genomic libraries usually contain large fragments and are likelyto be constructed in YAC or BAC vectors.

E)

cDNA libraries contain sequences from expressed genes only.

Which of the following is NOT a typical component of apolymerase chain reaction (PCR)?

A)

dNTPs

B)

DNA containing the sequences to be amplified

C)

DNA ligase to connect the fragments together

D)

Primers complementary to each end of the sequence to beamplified

E)

Heat stable Taq polymerase

How can a fusion protein be formed?

A)

Site-directed mutagenesis where a fragment is replaced with asynthetic sequence containing the altered DNA sequence.

B)

Two proteins can recombine in the bacterial cell to form a newhybrid protein.

C)

Portions of two different genes are ligated together to create anew hybrid gene. When expressed the gene product is a fusion of thetwo gene products.

D)

Using oligonucleotide-directed mutagenesis to introduce specificmutations that cause the expressed protein to fuse to otherproteins.

E)

A portion of the gene can be removed to form a shorter gene andtherefore a shorter protein.

Why is green fluorescent protein (GFP) so useful in visualizingfusion proteins in eukaryotes?

A)

The GFP protein recognizes and binds to fusion proteins allowingthe location of the fusion protein to be determined.

B)

The reaction is only dependent on the presence of GFP and oneother cofactor.

C)

It is easily cloned and expressed in bacterial cells.

D)

The presence of just a few molecules of GFP fusion proteins canbe sufficient for observing the proteins microscopically allowingthe study of its location and movements in the cell.

E)

It is derived from fire flies, which are easy to cultivate inthe lab.

You wish to determine the cellular location of a protein butunfortunately it is not particularly antigenic (can t develop astrong antibody response). How could you solve this problem?

A)

Create a fusion protein with an epitope tag that can bevisualized by incubation with fluorescently tagged antibody to theepitope.

B)

Create a fusion with green fluorescent protein (GFP).

C)

Express the protein as a fusion with biotin. The addition of GFPwill cause fluorescence.

D)

A and B might work.

E)

A, B, and C might work.

F)

None of the above will work.

A positive result for the yeast two-hybrid analysis means thefollowing:

A)

The proteins that you are studying can be made into fusionproteins, but do NOT interact with each other within the nucleus ofyeast.

B)

A fusion protein containing the Gal4p activation domain, hasbound to RNA polymerase resulting increase transcription ofnumerous genes, including the reporter gene, and formation ofcolonies.

C)

Two fusion proteins, one containing the Gal4p binding sitedomain and one containing the Gal4p activation domain, haveinteracted through their Gal4p domains resulting in transcriptionof the reporter gene.

D)

Two fusion proteins interact with each other and because of theGal4p binding site domain and the Gal4p activation domain containedin each fusion protein, the reporter genes are activated and thecells die resulting in no colonies.

E)

The colonies growing are expressing the two fusion proteins, butthe two proteins have not interacted with each other.

Which of the following is the approximate size of the humangenome?

A)

3 Gm

B)

300 Kb

C)

3 Mb

D)

3 Gb

E)

3 Mm

Which of the following is correct about the structure orlocation of genes?

Question 22 options:

A)

Prokaryotic genes typically possess their own regulatorysequences that only control expression of a single gene.

B)

Eukaryotic genes often encode introns that are spliced outbefore translation.

C)

Eukaryotic genes are mostly located at the telomeres and thecentromeres of chromosomes.

D)

Eukaryotic genes are typically arranged in operons.

E)

Prokaryotic genes often encode exons that are spliced out beforetranslation

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

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 92-95℃ for about one minute, to separate it into two single strands.

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

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

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