Lab1

Question 1

gene manipulation

Answer 1

the formation of new combinations of heritable material by the insertion of nucleic acid molecules produced by whatever means outside the cell, into any virus, bacterial plasmid or other vector system so as to allow their incorporation into a host organism in which they do not naturally occur but in which they are capable of continuing propagation

Question 2

“continued propagation” feature:

Answer 2

the foreign nucleic acid molecules mut be maintained in the host organism

Question 3

early experiments achieved “continued propagation” feature by

Answer 3

linking exogenous DNA covalently to autonomously replicating bacterial plasmids

Question 4

Two key points of gene manipulation

Answer 4

capacity to transgress natural biological barriers so genes from any organism can be transferred and maintained in an unrelated organism
- defined relatively small segments of DNA are propagated(breed) in the host organism

Question 5

consequences of gene manipulation

Answer 5

• intentional creation of novel genetic combinations by biochemical means
• perpetual propagation of such newly created lines of genetically identical organisms

Question 6

Each of the clones will contain foreign DNA which can ________
thereby
_______________

Answer 6

be expanded in bulk

thereby amplifying the foreign gene and the product specified(which forms the basis of modern biotechnology)

Question 7

Three techniques for gene manipulation(developed in early 1970’s)

Answer 7

• the transformation of Escherichia coli
• the capacity to cleave DNA molecules at precise and consistent points and to ligate these together to create novel arrangements of genetic material
• the capacity to monitor the cleaving and ligating of DNA molecules quickly and reliably

Question 8

cloning

Answer 8

the exercise of obtaining a group of organisms or cells of identical genetic composition that are descended from a commo ancestor by asexual reproduction

Question 9

One can refer to DNA or gene cloning as

Answer 9

the isolation and replication of a segment of DNA by laboratory manipulation

Question 10

Subcloning

Answer 10

moving a cloned gene from one vector to another

Question 11

Gene manipulation often requires

Answer 11

using pure DNA of known concentration

Question 12

Two common methods to assess DNA purity and quantity

Answer 12

UV spectrophotometry and fluorescenece of GelRed-bound DNA

Question 13

Otherwise colourless biomolecules can often be converted into _____________ by _____________ that permit __________.
This happens because of the capacity of various biomolecules to _________

Answer 13

coloured derivatives

• chromogenic reactions
• spectrophotometric analyis
• absorb light

Question 14

Conventional ultraviolet-visible spectrophotometry(which utilizes light absorption over the range of ________) is used for ___________ and/or for __________________

Answer 14

200-800 nm
the determination of purity
qualitative methods or quantitative estimates of biomolecules

Question 15

spectrophotometirc assessment by the extent of ultraviolet light absorbed by the bases can be performed if

Answer 15

the concentration of DNA is not too low (> 5µg/ml)

and the sample is pure

Question 16

To quantitate the amount of DNA (or RNA) in a sample, spectrophotometric readings should be taken at

Answer 16

both 260 and 280 nm

Question 17

The spectrophotometric value at 260 nm provides

Answer 17

an estimate of the concentration of the nucleic acid in the sample

Question 18

pure preparations of DNA and RNA have OD260nm/OD280nm values of
These values are lower when ______

Answer 18

1.8 and 2.0 respectively

The sample is contaminated with protein/phenol/any other UV-absorbing material

Question 19

Florescene of GelRed-Bound DNA often used when _____

As is often the case when ___________

Answer 19

the amount of DNA is low

A fragment generated from a restriction enzyme digest is isolated and purified

Question 20

Gelred

The intensity of the unkown band is

Answer 20

• enables direct visualization of the samples by a method of staining where it intercalates the bases of the double helix and when it is exposed to UV radiation it fluoresces in the visible range
• compared to the intensity of the marker band with a size closest to that of the unknown band

Question 21

Restriction endonucleases are _________

they yield a measure of __________

Answer 21

• a class of bacterial DNases involved in the recognition and destruction of foreign DNA that may enter a cell
• protection against bacteriophage infection and the transfer of genes between species

Question 22

Types of restriction enzymes used in the lab

they most commonly recognize __ bases but may recognize specific sequences between _______ bases

Answer 22

type 2
6 bases
4 and 12 bases

Question 23

Restriction enzyme nomenclature
First three letters -
the other letters

Answer 23

• italicized and refer to the genus and species of the bacteria where it is found
• refer to the numbering given to a particular enzyme

Question 24

restriction enzymes are the basic tool that

they are often used to

Answer 24

• allows DNA to be cut into reproducible fragments and moved from one place to another
• reduce large genes into smaller pieces suitable for sequencing and are used to generate physical maps of the DNA

Question 25

physical mapping is often the quickest way by which one can

Answer 25

verify the size and the orientation of DNA inserted into a plasmid

Question 26

DNA ligases covalently __________

catalyze the formation of ________ which constitues a ________

Answer 26

• join or splice together dna fragments of interest and the opened vector
• phosphodiester bonds between the 3’-hydroxyl and the 5’-phosphate groups of adjacent nucleotides
• nick in an otherwise intact DNA duplex

Question 27

There are two major variants of the ligation reaction used in gene manipulation

Answer 27

the joining of annealed cohesive ends produced by certain restriction enzymes or the joining of blunt ended fragments

Question 28

In the first step of any ligation reaction _______
This amp is trasnferred to ______ which thus ______
a phosphodiester bond forms when

Answer 28

• both enzymes form an enzyme-AMP intermediate with the AMP-coming from two different sources: ATP for the T4 enzyme and NAD+ for the bacterial enzyme
• the 5’-phosphate at the gap between the two DNA fragments
• activates the phosphate
• the 3’-hydroxyl group attacs the activated 5’-phophate moiety, eliminating free AMP

Question 29

blunt or cohesive ends can result in

Answer 29

exclusion of the insert by vector recircularization(or dimerization) or in inclusion of the insert but in one of two orientations

Question 30

vector recircularization and dimerization can be prevented by _______ which will remove ______

Answer 30

• pre-treating the vector with calf intestinal phosphatase

- the 5’ termini

Question 31

If non-coheisve ends are ligated, then the only possible result is
This is known as

Answer 31

• incorporation of the insert in one orientation

- directional cloning

Question 32

blunt ends are harder to

becuase there may be rejoined to

Answer 32

• rejoin

- any blunt end

Question 33

ligations should be done at high ______-

because

Answer 33

DNA concentrations

- dilute solutions favour circularization

Question 34

the ligases function best at
but the unligated cohesive end
so then the optimum temperature for a given ligation reaction is

Answer 34

37˚C
may not be stable at this temperature
a compromise between the rate of the enzyme reaction and the association of the termini

Question 35

Transformation describes

Answer 35

the means by which the novel combinations of DNA molecules created in vitro(test tube) are brought into the living organism

Question 36

what were created intentionally as “vehicles” for gene manipulation

Answer 36

bacterial plasmids containing an origin of replication and one of more antibiotic resistence genes

Question 37

Some plasmids exist at levels of a few copies per cell because

Answer 37

their replication is tightly coupled to replication of the host’s own chromosome

Question 38

When plasmids are said to be more relaxed it is because

Answer 38

plasmid replication is not coupled to chromasome replication, although it employs the DNA synthesizing machinery of the host
(these typically exist at levels of 10-20 copies per cell)

Question 39

It is possible to stop cell growth and chromosome replication without _______________
This is because ___________

Answer 39

• affecting plasmid replication

- chromosome replication is tightly coupled to cell growth

Question 40

antibiotics such as chloramphenicol or spectinomycin ________
So cells treated with one one these will _________
but because they already contain DNA sythesizing enzymes, ____________

Answer 40

stop protein synthesis while the ells are actively growing

• make no more chromosomal dna
• these cells will continue to produce more plasmid molecules for several hours

Question 41

purification of plasmid from cellular source requires

Answer 41

separating plasmid DNA from chromosomal DNA, as well as the removal of RNA, protein and other cellular components

Question 42

plasmid molecules can be extracted from cells under conditions in which ______
because they are ______
the supercoiled form is ________

Answer 42

they remain in supercoiled form
small
very resistant to denaturation of base pairs

Question 43

large chromosomal DNA molecules are _______

to yield _________

Answer 43

sheared during cell lysis

DNA that is non-circular and easy to denature