CMMB 461 Lecture Notes - Lecture 3: Celera Corporation, Sanger Sequencing, Complementary Dna

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rosefrog223

14 Sep 2018

School

University of Calgary

Department

Cellular, Molecular and Microbial Biology

Course

CMMB 461

Professor

Gordon Chua

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Document Summary

These processes require dna polymerase, primer, template dna and dntps. Dna de novo, it needs a primer sequence to initiate the synthesis. Dna polymerase adds the next nucleotide at the 3"- Adding nucleotides to the daughter strand requires energy, which is attained from the two phosphate groups. Dna polymerase cleaves from the end of the nucleotide. This releases enough energy to link the nucleotide to the 3" end of the growing dna strand. Dna sequencing by the chain termination method (sanger sequencing) Different size fragments are generated during dna synthesis depending on the location of ddntp incorporation/termination. Dntp has oh at the 3" end where the next nucleotide will be added. Ddntp has h at the 3" end, instead of an oh group, therefore dna polymerase cannot add a new nucleotide, and the sequencing process stops. Ddntp is an inhibitor of the replication reaction. Add all 4 types of dntp and the ddntp of whichever nucleotide should come next.

Related Questions

1.When a protein binds to DNA at a site defined by a particular nucleotide sequence, with which parts of the DNA does the protein primarily interact?

A. the phosphate and deoxyribose groups in the DNA backbone

B. the phosphate and deoxyribose groups in the DNA backbone, and hydrophobic interactions with the nucleotide bases

C. nucleotide bases within the major grooves of the DNA

D. nucleotide bases within the major and minor grooves of the DNA

E. none of the above

2. Which of the following proteins binds to DNA with sequence specificity?

A. POI Ill holoenzyme

B. DnaA

C. DnaB

D. Dam methylase

E. Both B and D

3. Which of the following is NOT a functional parallel among all known cellular systems of DNA replication?

A. Replication is initiated at specific sites

B. Replication is continuous on the leading strand and discontinuous on the lagging strand

C. DNA replication is semiconservative

D. Replication forks move away from the origin in one direction

E. Nucleases, polymerases and ligases replace RNA primers with DNA and seal the remaining nick

4. A DNA polymerase cannot synthesize a DNA strand de novo (from the beginning) from a mixture of dNTPs. Which of the following explains the role of the primer in the reaction promoted by this enzyme?

A. The primer is an initiation protein with attached nucleotides that binds to the template and to which new nucleotides are added to the 3 ' end.

B. The primer is an initiation protein with attached nucleotides that binds to the template and to which new nucleotides are added to the 5' end.

C. The primer is a preexisting strand of RNA or DNA to which new nucleotides are added to the 3' end.

D. The primer is a preexisting strand of RNA or DNA to which new nucleotides are added to the 5' end.

E. The primer is a preexisting strand of DNA synthesized by DNA pol I, prior to replicative DNA synthesis by DNA pol Ill.

5. Universal features of DNA polymerases include:

A. 5'-3' exonuclease function.

B. Formation of an 'closed' conformation of the polymerase when the template-nucleotide base pair is correct.

C. Use of a single magnesium ion to orient reacting molecules and assist in dissipation of the negative charge that develops during nucleotidyl transfer.

D. Alignment of conserved residues in the active site for attack of the DNA primer by the 3 'OH on the incoming nucleotide.

E. Continuous unbinding and rebinding of the polymerase to the DNA template as each nucleotide is added.

byzantiumscorpion425

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

31. Why does incorporation of a dideoxynucleotide into a growingDNA polymer terminate strand synthesis?

a. There is no longer a free 5âphosphate to which to add the next nucleotide.

b. There is no longer a free 3â hydroxyl to which to add thenext nucleotide.

c. There is no longer a free 3â phosphate to which to add thenext nucleotide.

d. There is no longer a free 5â hydroxyl to which to add thenext nucleotide.

2. The output from an automatedsequencing reaction shows a series of fluorescent peaks. Whenconsidering the data output, why are some peaks larger thanothers?

a. Chain termination occurred more frequently at the basecorresponding to a large peak, producing a relatively largerfluorescent signal.

b. The primer had multiple bases fluorescently labeled forproducts corresponding to the larger peaks.

c. More bases were fluorescently labeled in the DNA strandscorresponding to the larger peaks.

d. Some fluorescent colors are brighter than others.

3. What allows chain terminationto occur at each base of the template during an automatedsequencing reaction?

a. the presence of a relatively low concentration of ddNTPs

b. the presence of a relatively low concentration of dNTPs

c. the presence of a relatively high concentration of ddNTPs

d. the presence of a relatively high concentration of dNTPs

4. In automated fluorescent sequencing, what would happen if thefluorescently labeled ddATP was inadvertently omitted from thereaction?

a. DNA polymerase does not function without equal concentrationsof the four nucleotides

b there would be gaps in the sequence where thymines werepresent in the template DNA strand

c. the sequence would end where the first thymine wasencountered on the template DNA strand

d. the sequence would end where the first adenine should beincorporated

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CMMB 461 Lecture Notes - Lecture 3: Celera Corporation, Sanger Sequencing, Complementary Dna

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