Chapter 3 Flashcards
5’ –> 3’ exonuclease
cleaves DNA in the 5’ to 3’ direction, this activity is required to remove RNA primers for DNA synthesis
3’ –> 5’ exonuclease
cleaves DNA in the 3’ to 5’ direction; this activity is important for “proofreading” DNA synthesis
Endonuclease
an enzyme that cleaves a DNA strand internally
Bacteria produce two different enzymes to protect its genome
1) Site specific methylase - enzyme methylates specific sequences of DNA in bacterial genome
2) Restriction endonulcease - enzyme cleaved specific DNA sequence; however does not cleave ow DNA, because DNA is methylated by site-specific methylase
Restriction endonuclease
enzyme cleaves specific DNA sequence; however does not cleave own DNA because DNA is methylated by site-specific methylase
Type I restriction enzyme
Contain both methylase/restriction endonuclease activities
Methylate DNA at specific sites
Cleave DNA 400-1,000 nucleotides from recognition site
Type II restriction enzyme
Restriction endonuclease activity only
Do not methylate DNA
Cleave DNA within or within a few nucleotides of recognition site - sequence is usually palindromic
Type of restriction enzyme typically used for cloning
Often work as a dimer
Type III restriction enzyme
Contain both methylase/restriction endonuclease activities
Methylate DNA at specific sites
Cleave DNA 20-30 nucleotides from recognition site
5’ Overhang
strands are cleaved asymmetrically so that overhanging ends contains 5’ P; refereed to as a sticky end
3’ Overhang
strands are cleaved asymmetrically so that overhanging ends contains 3’ OH; refereed to as a sticky end
Blunt
strands are cleaved symmetrically so that there is no overhang
Isoschisomers
different restriction enzymes that can recognize the same sequence and cleave in the same location
Neoschizomers
different restriction enzymes that can recognize the same sequence but cleave in different locations
Factors influencing restriction enzyme efficiency
1) Buffer conditions - different restriction enzymes have different preferences for ionic strength and major cation
2) Temperature - most restriction enzymes work best at 37C, some exceptions
3) Methylation - some restriction enzymes will not cleave methylated DNA
4) ‘Star’ activity = cleavage in nonstandard conditions; this can lead to cleavage of sequences that differ from the normal recognition site by only a basepair
Blunting DNA
1) fill in of 5’ overhangs using DNA polymerase and dNTPs
2) Removal of 3’ overhangs using nucleases (e.g., Mung bean nuclease
DNA ligase
Absolutely essential for phosphodiester bond formation between 3’ OH and 5’ P. Ligates the pieces of DNA together
Components necessary for genomic DNA replication
1) Helicase - unwinds double-stranded DNA
2) Single-stranded binding protein - binds single stranded DNA formed by unwinding strands
3) Primase - synthesizes short RNA primer with free 3’ OH
4) DNA polymerase III - synthesizes DNA from RNA primers; has ‘proofreading’ activity
5) Topoisomerases - necessary to remove supercoiling formed by unwinding strands
6) DNA polymerase I - has exonuclease activity to remove RNA primers and fill in the gap
7) DNA ligase - necessary to form phosphodiester bond between adjacent nucleotides
Requirements for in vitro DNA replication
1) DNA template - need to have genetic information in which to duplicate
2) Free 3’ OH group - DNA synthesis cannot occur without the attachment of new nucleotides to the 3’ OH group of the preceding nucleotide. This requirement is met by using synthetic primers.
3) Deoxynucleotide triphosphates (dNTPs) - the building blocks of DNA
4) DNA polymerase - need enzyme to add ‘building blocks’ to the growing strand of newly synthesized DNA
5) Mg2+ - magnesium is a necessary cofactor for DNA polymerase
Polymerase chain reaction (PCR)
in vitro DNA synthesis by Kary Mullis
Synthesis is repeated for 30-40 cycles to amplify a specific region of DNA
1 PCR cycle:
1) Denaturation - DNA is heated (92-95C) to separate DNA strands
2) Annealing - DNA is cooled (50-65C) to allow primers to anneal to DNA
3) Extension - DNA is heated (68-72C) to allow for optimal synthesis by polymerase
Exponential amplification of DNA
(2^n -2(n))
Processivity
rate at which the polymerase copies the template
Fidelity
accuracy of the template copy being made
Persistence
the stability of the enzyme at high temperature