Biotechnology (W16) - Recombinant DNA technology Flashcards
The ability of researcher’s to manipulate DNA at the molecular level is known as _________ (more than one word).
Recombinant DNA technology or genetic engineering
The term _________ ___ refers to the ability to prepare novel DNA species by recombining DNA from different sources.
recombinant DNA
Most of the tools of recombinant DNA technology have arisen from __________ and _______.
bacteriophage and bacteria
______ are small, circular double-stranded DNA molecules that are common in bacteria and which have been adapted to become key ______ for recombinant DNA.
1 - Plasmids
2 - vector
_______ ________ are enzymes that cleave dsDNA at specific sites called _________ _________.
1 - Restriction endonucleases
2 - recognition sites
Bacteria use _________ __________ as a defense mechanism.
restriction endonucleases
In recombinant DNA technology, _________ __________ are used as molecular scissors to cleave ____ molecules to allow them to be recombined with other ____ molecules.
1 - restriction endonucleases
2 - DNA
3 - DNA
DNA and RNA polymerases synthesize nucleic acids in a __________-dependent manner.
template
DNA ligases catalyze the formation of ____________ bonds to allow assembly of _________ ____ constructs.
1 - phosphodiester bonds
2 - recombinant DNA
What is cDNA?
Double stranded DNA copies of RNA
Expression of proteins using recombinant technology affords the ability to: (2 things)
1 - ovexpress proteins for the purpose of purifying them
2 - selectively mutate one or more residues in the protein for the purposes of structure/function analyses of the protein
A technique known as _____-__________ __________ is performed to introduce the desired mutations into the cloned cDNA and is used to alter nucleotide sequence of cloned DNA at a predefined position or site.
site-directed mutagenesis.
SDM can be used to change only a single or multiple _____-_____, or can be used to create more extensive sequences changes such as ______ and ________ or ______ or ________ of restriction enzyme sites.
1 - base-pairs
2 - insertions and deletions
3 - removal or introduction
There are two general methods for oligonucleotide site-directed mutagenesis. What are they?
1 - Synthetic DNA casettes
2 - Enzymatic extensions of a mutagenic oligonucleotide (also called SDM)
What is the end result of both methods of oligonucleotide SDM?
an oligonucleotide encoding the new DNA sequence is inserted into a cloned DNA fragment
What method will we be using?
Enzymatic extension of a mutagenic oligonucleotide
The DNA polymerases used for the method we are using are?
Bacteriophage T4 or R7.
Why are we using bacteriophage T4 or R7?
Do not proofread mutagenic oligonucleotides therefore making it possible to obtain mutant plasmids
mutant DNA is also called?
heteroduplex DNA
How is the system able to remove the template DNA prior to bacterial transformation?
pcr product is digested with the endonuclease Dpn1
What does the endonuclease Dpn1 do?
digests methylated sequences in the template DNA
The sequence of interest (the newly created mutant DNA) is transformed into competent cells which are able to perform _____ ______.
nick repair
Draw the SDM process.
Refer to notes.
1 - template DNA (plasmid vector) is denatured to become ss
2 - anneal mutagenic oligonucleotide to ss template DNA
3 - Add DNA pol (T4/R7), DNA ligase, ATP and dNTPs
4 - Synthesis of complementary strand
5 - DNA ligase joins end, have heteroduplex DNA (mixture of both wt and mutant DNA)
6 - Remove parental DNA by Dpn1 methylated DNA digestion
7 - Transform into competent cells that perform nick-repair
The wt sequences need to be long enough so a stable DNA duplex can form between the ___________ _____ and the template DNA.
oligonucleotide primer
