Basic Cloning Methodologies Flashcards
(37 cards)
What is cloning?
Production of a group of IDENTICAL cells or organisms
What is an example of “natural” clones?
Identical Twins
What is cloning in molecular biology?
Placing a foreign gene in bacterial cells, isolating individual cells, then growing colonies from the individual bacterial cells. All the cells in the bacterial colony (batch culture) will be identical and contain the foreign DNA
When and by who was the first cloning experiment done?
Stanley Cohen, Herbert Boyer, and colleagues in 1973
What is a critical part of cloning experiments and by who was this discovered?
Restriction endonucleases
Linna and Arber late 60s
Why are restriction enzymes called restriction enzymes?
They prevent the invasion of foreign viral DNA into bacterial cells, thus restricting the host range of the virus. The invading viral DNA is cut into pieces by the restriction endonuclease.
How specific were the enzymes discovered by Linn and Arber regarding the sites of DNA cleavage?
NOT specific at all
The endonuclease discovered by Smith and used by Cohen and Boyer was isolated from what organism and what made it different from others?
The enzyme was isolated from Haemophilus influenza, strain Rd and it was an endonuclease that cleaved SPECIFIC sites in the DNA.
How are the names of restriction enzymes derived?
And give an example.
From the Latin name of the organism that produces them. The first letter of the enzyme is the first letter of the genus name. The next two letters come from the first two letters of the species name. The strain name is added as plus the number indicating the sequential number of nucleases produced.
Example: Hind II from the organism Haemophilus influenza
What is the sequence recognized by Hind II?
GTPyPuAC
CAPuPyTG
where Py is either T or C and Pu is either A or G
What three categories of cutters can restriction endonucleases fall into and what are their commonalities?
4-cutters (frequent cutters)
6-cutters (common)
8-cutters (rare cutters)
How often do 4-cutters cut?
once every 256 base pairs
How often do 6-cutters cut?
once every 4096 base pairs
How often do 8-cutters cut?
once every 65,000 base pairs
Restriction enzymes cut what type of DNA?
double stranded DNA
WHat are two or more restriction endonucleases called that recognize the same restriction site and cut at the same location?
Isoschizomers
What are two or more restriction endonucleases called that recognize the same restriction site but cut at different locations?
Neoschizomers
What are blunt ends?
When restriction endonucleases cut straight across DNA and leave straight ends
What are sticky ends?
When restriction endonucleases cut DNA and eave staggered edges and overhangs
WH
hat are palindromic sequences, give an example, and what do they typically result in?
Sequences that have two fold symmetry and are identical after roatating the sequence 180 degrees (i.e. frontwards and backwards)
Example: 5’-GAATC-3’
3’-CTAAG-5’
They result in sticky ends.
WHat is the R-M System or the Restriction Modification System?
A system in which a methylase is almost always paired with a restriction endonuclease and the methylase protects the host DNA and allows the restriction endonuclease to chop up only the viral DNA.
How does the restriction modification system work?
The methylase recognizes the same base sequence as its corresponding endonuclease. The methylase methylates a base in the recognition sequence by adding a methyl group -Ch3. The presence of this methyl group protects the DNA.
If restriction endonucleases chop up DNA, why don’t they chop up the HOST DNA and not just the invading viral DNA?
Host DNA is methylated and viral DNA is unmethylated
What is Hemi-methylation?
It protects newly synthesized DNA from endonucleases during replication
