Lab1 Flashcards
gene manipulation
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
“continued propagation” feature:
the foreign nucleic acid molecules mut be maintained in the host organism
early experiments achieved “continued propagation” feature by
linking exogenous DNA covalently to autonomously replicating bacterial plasmids
Two key points of gene manipulation
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
consequences of gene manipulation
- intentional creation of novel genetic combinations by biochemical means
- perpetual propagation of such newly created lines of genetically identical organisms
Each of the clones will contain foreign DNA which can ________
thereby
_______________
be expanded in bulk
thereby amplifying the foreign gene and the product specified(which forms the basis of modern biotechnology)
Three techniques for gene manipulation(developed in early 1970’s)
- 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
cloning
the exercise of obtaining a group of organisms or cells of identical genetic composition that are descended from a commo ancestor by asexual reproduction
One can refer to DNA or gene cloning as
the isolation and replication of a segment of DNA by laboratory manipulation
Subcloning
moving a cloned gene from one vector to another
Gene manipulation often requires
using pure DNA of known concentration
Two common methods to assess DNA purity and quantity
UV spectrophotometry and fluorescenece of GelRed-bound DNA
Otherwise colourless biomolecules can often be converted into _____________ by _____________ that permit __________.
This happens because of the capacity of various biomolecules to _________
coloured derivatives
- chromogenic reactions
- spectrophotometric analyis
- absorb light
Conventional ultraviolet-visible spectrophotometry(which utilizes light absorption over the range of ________) is used for ___________ and/or for __________________
200-800 nm
the determination of purity
qualitative methods or quantitative estimates of biomolecules
spectrophotometirc assessment by the extent of ultraviolet light absorbed by the bases can be performed if
the concentration of DNA is not too low (> 5µg/ml)
and the sample is pure
To quantitate the amount of DNA (or RNA) in a sample, spectrophotometric readings should be taken at
both 260 and 280 nm
The spectrophotometric value at 260 nm provides
an estimate of the concentration of the nucleic acid in the sample
pure preparations of DNA and RNA have OD260nm/OD280nm values of
These values are lower when ______
1.8 and 2.0 respectively
The sample is contaminated with protein/phenol/any other UV-absorbing material
Florescene of GelRed-Bound DNA often used when _____
As is often the case when ___________
the amount of DNA is low
A fragment generated from a restriction enzyme digest is isolated and purified
Gelred
The intensity of the unkown band is
- 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
Restriction endonucleases are _________
they yield a measure of __________
- 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
Types of restriction enzymes used in the lab
they most commonly recognize __ bases but may recognize specific sequences between _______ bases
type 2
6 bases
4 and 12 bases
Restriction enzyme nomenclature
First three letters -
the other letters
- italicized and refer to the genus and species of the bacteria where it is found
- refer to the numbering given to a particular enzyme
restriction enzymes are the basic tool that
they are often used to
- 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
physical mapping is often the quickest way by which one can
verify the size and the orientation of DNA inserted into a plasmid
DNA ligases covalently __________
catalyze the formation of ________ which constitues a ________
- 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
There are two major variants of the ligation reaction used in gene manipulation
the joining of annealed cohesive ends produced by certain restriction enzymes or the joining of blunt ended fragments
In the first step of any ligation reaction _______
This amp is trasnferred to ______ which thus ______
a phosphodiester bond forms when
- 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
blunt or cohesive ends can result in
exclusion of the insert by vector recircularization(or dimerization) or in inclusion of the insert but in one of two orientations
vector recircularization and dimerization can be prevented by _______ which will remove ______
- pre-treating the vector with calf intestinal phosphatase
- the 5’ termini
If non-coheisve ends are ligated, then the only possible result is
This is known as
- incorporation of the insert in one orientation
- directional cloning
blunt ends are harder to
becuase there may be rejoined to
- rejoin
- any blunt end
ligations should be done at high ______-
because
DNA concentrations
- dilute solutions favour circularization
the ligases function best at
but the unligated cohesive end
so then the optimum temperature for a given ligation reaction is
37˚C
may not be stable at this temperature
a compromise between the rate of the enzyme reaction and the association of the termini
Transformation describes
the means by which the novel combinations of DNA molecules created in vitro(test tube) are brought into the living organism
what were created intentionally as “vehicles” for gene manipulation
bacterial plasmids containing an origin of replication and one of more antibiotic resistence genes
Some plasmids exist at levels of a few copies per cell because
their replication is tightly coupled to replication of the host’s own chromosome
When plasmids are said to be more relaxed it is because
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)
It is possible to stop cell growth and chromosome replication without _______________
This is because ___________
- affecting plasmid replication
- chromosome replication is tightly coupled to cell growth
antibiotics such as chloramphenicol or spectinomycin ________
So cells treated with one one these will _________
but because they already contain DNA sythesizing enzymes, ____________
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
purification of plasmid from cellular source requires
separating plasmid DNA from chromosomal DNA, as well as the removal of RNA, protein and other cellular components
plasmid molecules can be extracted from cells under conditions in which ______
because they are ______
the supercoiled form is ________
they remain in supercoiled form
small
very resistant to denaturation of base pairs
large chromosomal DNA molecules are _______
to yield _________
sheared during cell lysis
DNA that is non-circular and easy to denature
