3 - Molecular Cloning Methods Flashcards
Learn slides from chapter 3 (30 cards)
In what year was the first sheep cloned
1996 cloned but born 1997
What three steps are involved with the cloning process?
Place a foreign gene into bacterial cells
Separate individual cells
Grow colonies from each of them.
What year was the first cloning experiment?
1973
Linn and Arber discovered restriction endonucleases in ____ in the late ____.
Linn and Arber discovered restriction endonucleases in E. coli in the late 1960.
Why are Restriction endonucleases named that way?
They restrict the host range of virus
They cut at sites within the foreign DNA, rather than chewing it away at the ends.
An Enzyme from ______ _______ Rd was the first enzyme to show specificity in cutting DNA.
An Enzyme from Haemophilus influenzaestrain Rd was the first enzyme to show specificity in cutting DNA.
How are Restriction Endonucleases designated?
Restriction enzymes derive the first three letters of their names from the Latin name of the microorganism that produces them:
First letter is the first letter of the genus H
The next two letters are the first two of the species
In some cases the strain designation is included, in this case d
Regarding Restriction Endonucleases, What is a rare cutter?
A restriction endonuclease that recognizes 8-bp(example NotI) therefore cut much less frequently
What are Heteroschizomers?
Heteroschizomers (Greek: hetero = different; schizo = split) or neoschizomers are enzymes that recognize different sites in identical sequences
What are Isoschizomers?
Isoschizomers (Greek: iso= equal) are enzymes that cut at the same site in the same sequence.
What is a palindrome sequence?
A palindrome sequences is a sequence with twofold symmetry. They read the same forward and backward.
What two types of cuts are possible with Restriction endonucleases?
Blunt - Flat cut leaving blunt ends
Single strand overhang - staggered cuts in the two DNA strands
If endonucleases can cut invading DNA, why don’t they destroy the host cell’s own DNA?
Methylase enzymes recognize and methylate the same DNA sites. Once methylated, DNA sites are protected against most endonucleases.
Almost all restriction enzymes are paired with a methylase enzyme, that recognizes and methylates the same DNA sites. what is this system called?
This system is called the R-M system (restriction-modification system)
Restriction enzymes are classified biochemically into what three types?
Restriction enzymes are designated as Type I, Type II, Type III.
A major type of Type II enzymes are sometimes referred to as ______ enzymes.
A major type of Type II enzymes are sometimes referred to as Type IV enzymes.
Type I and III systems are unique how?
Type I and III systems, both the methylase and restriction activities are carried out by a single large enzyme complex.
True or False:
Type I and III systems Cut adjacent to the recognition site
False
Type I and III enzymes recognize specific DNA sequences, but the sites of cleavage are at variable distances from the recognition sites, and can even be hundreds of bases away.
What is required for all three classifications?
ATP
How is Type II restriction enzymes unique?
In type II systems, the restriction enzyme is independent of its methylase, and cleavage occurs at very specific sites that are within or close to the recognition sequence.
What of the three classifications is the most common? (Type I, II, or III)
The vast majority of known restriction enzymes are of type II.
How are Type II enzymes further classified according to their recognition site?
Type II
Type IIa
Type IIb
Type IIs
How do type II enzymes cut?
Most type II enzymes cut palindromic DNA sequences
How do type IIa enzymes cut?
Type IIa enzymes recognize non-palindromic sequences and cleave outside of the recognition site
