Methods 1 (lecture 3) DNA replication techniques

Question 1

To estimate the Tm of an oligonucleotide?

Answer 1

(#A + #T) x 2oC + (#G + #C) x 4oC = Tm of a primer

Question 2

steps of bacterial plasmid DNA extraction?

Answer 2

1. Grow bacteria overnight
   2.Collect cells by centrifugation
2. Resuspend the cells in a buffer (contains glucose to maintain osmolarity, EDTA to chelate metal ions, de-stabilize membrane proteins, and inactivate DNases, and Tris to maintain pH)
3. Lyse the cells with SDS (a detergent that makes holes in the membranes) plus NaOH (loosens cell walls, denatures the proteins, releases and denatures DNA)
4. Neutralize the solution with potassium acetate that converts SDS to insoluble KDS: at neutral pH plasmid DNA re-anneals, whereas sheared chromosomal DNA remains single stranded and insoluble at high salt
5. Centrifuge the sample to get rid of cellular debris, denatured proteins, KDS, and ssDNA chromosomal DNA
6. The supernatant that contains plasmid DNA can then be either passed through a silica membrane (that will bind plasmid DNA under high salt conditions) or precipitated with alcohol and centrifuged to collect the DNA on the bottom of the tube
7. Plasmid DNA (on a membrane or in a pellet) is washed with 70% ethanol
8. Pure DNA (eluted from the membrane under low salt conditions or resuspended from a pellet) can then be used for downstream applications (PCR, restriction digest, etc.)

Question 3

What is a multiple cloning site

Answer 3

Site in a plasmid contains recognition sequences for several different restriction enzymes

Question 4

What is a restriction enzyme

Answer 4

enzymes which recognize a specific, usually palindromic, sequence (typically 4, 6 or rarely 8 bp long) in a plasmid

Question 5

potential components of a bacterial plasmid

Answer 5

1. multiple cloning site
2. reporter for selection, can be interrupted by multiple cloning site
3. origin of replication (controls copy number of plasmids)
4. selectable marker/antibiotic resistance gene (select bacteria or other cells containing plasmid)

Question 6

restriction enzymes bind ds or ss DNA?

Answer 6

dsDNA

Question 7

what bonds do restriction enzymes cut?

Answer 7

phosphodiester

Question 8

plasmid DNA becomes ___ after cutting?

Answer 8

linearized

Question 9

if restriction enzymes cut plasmid in multiple places you generate ___

Answer 9

multiple dsDNA fragments

Question 10

Restriction enzymes make 3 different types of cuts, what are they?

Answer 10

5’ overhangs, 3’ overhangs, blunt

Question 11

what enzyme is used to re-attach blunt or sticky ends resulting from restriction enzyme cutting a plasmid?

Answer 11

DNA ligase

Question 12

what are “sticky ends”?

Answer 12

staggered DNA ends with sequences complementary to one another

Question 13

Would you use the same restriction enzyme or different to produce sticky ends with sequence similarity?

Answer 13

You could use one enzyme or two different enzyme to cut complimentary sites in two separate DNA molecules, the resulting sticky ends can be ligated by DNA ligase

Question 14

What is subcloning?

Answer 14

cutting and ligating desired pieces of DNA together in a vector using a combination of restriction enzymes and DNA ligase

Question 15

what is “competence” in regards to cloning?

Answer 15

competence is the ability of bacteria to accept foreign DNA (the ability to accept a plasmid)

Question 16

two methods to make bacteria competent??

Answer 16

chemically- suspend in CaCl2 solution (permeabilize membrane) and subject cells to mild heat shock (make cells uptake DNA in a survival attempt) then allow recovery before antibiotic selection

electrically- wash to remove salts (ions will cause cell explosion essentially) and subject to short electric pulse

electroporation is more efficient than chemical competance

Question 17

Why do bacteria maintain and not kick out plasmid?

Answer 17

plasmids impart antibiotic resistance (antitoxin to antibiotic)

Question 18

reporter genes are often inserted where in a plasmid?

Answer 18

In the multiple cloning site

Question 19

example of reporter genes used in plasmids?

Answer 19

LacZ code for b-galactosidase

b-galactosidase converts a substrate into a blue product

Question 20

If a plasmid was inserted into a MCS containing LacZ would that colony be blue or white in the presence of the appropriate substrate?

Answer 20

If the plasmid was correctly inserted the substrate would stay white (as the LacZ gene would be interupted)

If plasmid was incorrectly inserted the colony would turn blue as you still have a functional LacZ gene

Question 21

Describe the origin of replication of a plasmid (simply)

Answer 21

an AT-rich region of DNA where replication starts

Question 22

If plasmid replication depends on host replication machinery is plasmid replication dependent or independent of host cell cycle?

Answer 22

dependent

Question 23

If plasmid replication depends on plasmid-encoded replication factors (Rep proteins) is plasmid replication dependent or independent of host cell cycle?

Answer 23

independent

Question 24

define replicon (in a plasmid)

Answer 24

origin of replication + all of its control elements

Question 25

How do different origins or replication (ORIs) result in different copy numbers of plasmids?

Answer 25

Depending on how they are regulated you can have just a few to hundreds of copies of a plasmid (“relaxed” or “stringent” control) depending on whether the ori is positively regulated by RNA or proteins, respectively. A plasmid’s copy number has to do with the balance between positive and negative regulation and can be manipulated with mutations in the replicon (mutations in the control elements)

Question 26

3 possible tests to confirm id of recombinant plasmid?

Answer 26

1. restriction digest DNA from selected colony + gel electrophoresis
2. PCR using gene specific primers
3. sequence the insert and its junction with the vector

Question 27

explain how restriction digest can be used to confirm id of recombinant plasmid?

Answer 27

1. digest the plasmid with a restriction enzyme
2. run the digested fragments on a gel
3. you know the size of the correct fragment, there should be 2 bands of the correct size if sequence was inserted

Question 28

How can one test the orientation of the insert in the plasmid by restriction analysis?

Answer 28

choose an enzyme/pair of enzymes to cut vector asymmetrically in the insert (cut the inside the insert + cut next to the insert in the vector), depending on the orientation of the insert you will get fragments of different sizes

Question 29

explain how PCR can be used to confirm id of recombinant plasmid?

Answer 29

design primer that overlaps both the vector + the insert, the primer will only allow amplification is the insert is oriented correctly

(either forward vector primer and reverse insert specific primer or vice versa)

Question 30

why is it important to orient insert correctly in plasmid?

Answer 30

Promoter must be upstream of insert to create the correct protein, or else you will make non-coding RNA (this noncoding RNA will be antisense and lead to RNAi mediated knockdown of gene of interest)

Question 31

describe 4 subcloning scenarios (is regards to the ends of insert/vector cut)

Answer 31

1. clone with 2 sticky ends
2. clone with 2 different but compatible ends (use different restriction enzymes that give you compatible ends)
3. clone with one sticky end and one blunt end (the ligation of the blunt end will be less efficient)
4. clone with 2 blunt ends (compatible ends have inefficient ligation and directionality is cloning)

Question 32

how to prevent self re-ligation of vector?

Answer 32

use alkaline phosphatase to de-phosphorylate the compatible vector ends, no p = no ligation. The insert will contain P on the 5’ ends so will be allowed to ligate, the 3’ end of the vector won’t ligate initially so there will be a nick (“nicked duplex”), the nick will be repaired by the bacteria