Manipulating Nucleic acids
When in DNA do restriction enzymes cleave at?
Cleavage sites: specific base sequences called palindromes (inverted repeats)
How can restriction enzymes help create recombinant DNA molecules?
By creating "sticky ends" in 2 different DNA molecules, which can then anneal with each other via complementary sticky ends.
Restriction enzyme recognition sites are of __________ bases in length
4 - 8
A four base site occurs every ________ bases.
A six base site occurs every _________ bases.
An eight base recognition sequence produces fragments between _____________ and _____________ bp in length
1 million - 1.5 million
The top of the electrophoresis gel where DNA is placed is ____________ charged
What can be used to visualize the position of DNA fragments in a gel? How does it work?
Ethidium bromide (fluorescent dye) intercalates between the DNA bases in dsDNA, and is illuminated with UV light
What does a Sanger sequencing reaction consist of?
1. A DNA strand to be sequenced
2. A short pieced of DNA primer that is complementary to the 3' end of the strand to be sequenced
3. Correct ratio of ddNTP/dNTP (the 4 ddNTPs can be labelled with different color fluorescence dye)
3b. (or label primer with 5' P32)
4. DNA polymerase
What are 2 ways of synthesizing oligonucleotides?
1) Use a chemical blocking group to block either the 5' or 3' end of a nucleotide. Allow a 5'-blocked and a 3'-blocked NT to react with each other. Remove one of the blocks with acid or base. Repeat.
2) Phosphite Triester Method
- perform with 3'end linked to insoluble support
- synthesize from 3' to 5' direction
In Phosphite Triester Method, what is used to block the 5'-end?
What catalyzes the coupling of the 3'-OH of a NT to the 5'OH of the growing chain in Phosphite triester method?
How is any uncoupled 5'OH prevented from reacting at later stages in Phosphite triester method?
blocked permanently by acetylation
What is the general cycle of synthesis in phosphite triester method?
After n cycles of PCR, how many dsDNA molecules that are copies of the DNA sequence between the primers will be produced?
How often do errors occur in naturally replicating DNA?
1 in 1 billion bp
How often do errors occur when using Taq polymerase in PCR?
1 in 20 000 bp
What are the 5 basic steps of cloning?
1) Section of DNA
2) Cut DNA with restriction enzyme
3) Mix with cut vector and ligate (splicing)
4) Introduce rRNA into bacterial host cells (transformation)
5) Isolate colonies (by plating bacteria on agar with antibiotic; vectors contain antibiotic-resistant genes)
cDNA is synthesized by what enzyme?
RNA-dependent DNA polymerase (reverse transcriptase)
cDNA may be _________ or _____________ stranded
What acts as the primer for reverse transcriptase when synthesizing cDNA?
Oligo(dT) that was used to purified the mRNA via the poly(A) tails
What can be attached to the Oligo(dT) primers so that directional cloning can occur later?
restriction sites (ie. Xhol) can be attached to the 5' end, to create sticky ends later
What cleaves the mRNA once the cDNA strand has been produced? What is produced as a result of this enzyme?
Fragments of mRNA, which are used as primers for E.coli DNA polymerase to produce a discontinuous strand of complementary cDNA.
For directional cloning, the resulting cDNA that is to be inserted into the vector has a sticky __________ site at the 5'end, and a sticky __________ site at the 3'end.