Biology 1202B Lecture Notes - Shotgun Sequencing, Southern Blot, Polyacrylamide Gel Electrophoresis

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ochrebee715

30 Apr 2012

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Polymerase chain reaction (pcr) - used to amplify something of interest in our dna: denature dsdna to ssdna, anneal dna primer to it. No end point of extension (no boundaries) just goes. You started with 1 double stranded dna, and end up with 2 double stranded. Some boundaries for end points: cycle 3: 2/8 dsdna defining the specific fragment of interest (i. e. primers are defining the ends) Insertion of dideoxyribonucleotides (does not have a 3" oh, only a hydrogen) stops synthesis: dntp and ddntp are being added randomly. If dntp is added, dna synthesis (sequencing) can continue. If ddntp is added, dna synthesis (sequencing) is stopped: creates fragments of various sizes, sequencing reaction is loaded into denaturing (ssdna) polyacrylamide gel with the ability to resolve fragments 1 nucleotide different in length. Polyacrylamide gel instead of agrose gel high resolution and single strands. 4 different fluorescent dyes, one for every ddntp.

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

The PCR (polymerase chain reaction) protocol that is currently used in laboratories was facilitated by the discovery of a bacterium called Thermus aquaticus in a hot spring inside Yellowstone National Park, in Wyoming. This organism contains a heat-stable form of DNA polymerase known as Taq polymerase, which continues to function even after it has been heated to 95Â°C. Why would such a heat-stable polymerase be beneficial in PCR?

What would happen if it werenât heat stable?

How might you choose a region of DNA for a PCR primer so as to increase the temperature necessary for primer annealing (to minimize nonspecific PCR products)?

A PCR reaction begins with 5 double stranded segment of DNA. Estimate the number of double-stranded copies of DNA that are present after the completion of 15 amplification cycles?

4) The polymerase chain reaction allows geneticists to make lots of copies of a particular strand of DNA rapidly. What are the purposes of the given PCR ingredients, and what are the ingredients whose purposes are given?

a. Taq DNA polymerase (2 pts)

b. __________________________are complementary to the 3' (three prime) ends of each strand of the initially denatured DNA target. These allow the polymerase to proceed in a 5â->3â direction. (2 pts)

c. _______________________________provides a solvent and Mg for the enzymatic reactions. (2 pts)

d. DNA nucleotides (2 pts)

e. Double stranded DNA template containing the _______________________________. (2 pts)

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Biology 1202B Lecture Notes - Shotgun Sequencing, Southern Blot, Polyacrylamide Gel Electrophoresis

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