BIOL 142 Lecture Notes - Lecture 22: Molecular-Weight Size Marker, Restriction Enzyme, Genetic Linkage

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28 Mar 2018

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Biol142 3/23/18 lecture 22, recombinant dna technology iii: gel electrophoresis. In the picture below, the underlined dna is the sequence that you want to amplify. Once the double-stranded dna is denatured (strands separate), primers will anneal: If you use an annealing temperature that is too high, hydroge(cid:374) (cid:271)o(cid:374)ds wo(cid:374)"t (cid:271)e a(cid:271)le to form between the dna primers and the template dna. Without the annealing step, pcr produ(cid:272)t (cid:272)a(cid:374)"t (cid:271)e for(cid:373)ed. It also is used to make the solution visible in the gel: a fluorescent dye like ethidium bromide may be used. It is an intercalating agent that inserts itself into structure of dna. Smaller dna fragments will migrate more easily (and thus faster), so they will travel a greater distance in a given amount of time: the fragments migrate as bands. Each band represents many molecules of a specific length of dna. Their common size allows them to migrate together: look at migration of bands under uv light, example:

Related Questions

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.

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

1. In a successful PCR reaction, which of the following is true?

a. The template DNA is added in excess of the primers and dNTPs.

b. The annealing temperature must be just below the melting temperature of the primers.

c. The temperature for the denaturation step is 72 C

d. All of the above

2. In our TLC experiment, which of the following had an effect on the migration of the samples?

a. The number of negatively charged phosphate groups

b. The protonation state of ionizable groups on the bases.

c. The size of the molecule.

d. All of the above

3. Which of the following is true of nucleic acid migration in agarose gel electrophoresis?

a.Nicked circular plasmids migrate faster than supercoiled plasmids.

b.Binding of ethidium bromide during electrophoresis speeds up the movement of linear fragments.

c.A higher percentage of agarose will decrease the migration of larger DNA fragments.

d.A lower strength electric field will increase the migration of DNA fragments.

4.What is the purpose of the outgrowth period during transformation?

a.To allow for the cells to take up DNA.

b.To allow for expression of the lacZÎ± and lacZÏ genes.

c.To allow for expression of antibiotic-resistance genes.

d.To prepare cell membranes for electroporation.

5.Which of the following is true of ligase and ligation reactions?

a.Ligase facilitates H-bonds between the sticky ends created by restriction enzyme digestion.

b.Ligase is inactivated at 37 C.

c.DNA fragments must have a 5â PO₄ group for ligase to covalently join them together.

d.Vector must be added in molar excess to insert to favor recombinant plasmid formation.

BIOL 142 Lecture Notes - Lecture 22: Molecular-Weight Size Marker, Restriction Enzyme, Genetic Linkage

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