Topic 7 Flashcards
(45 cards)
Which of the following repair mechanisms does not make use of a polymerase?
Direct repair
Nuclear excision repair
Mismatch repair
Non-homologous end joining
None of the above
Direct repair
What are the recombinant DNA techniques used to manipulate DNA?
Cloning: synthesize (polymerize) thousands of DNA copies
PCR: detect DNA fragments
Gel electrophoresis: quantify DNA fragments
Southern blot: retrieve (purify) DNA fragments
Define gene cloning
Introducing a desired DNA molecule that can be introduced and propagated in vivo inside a host cell, then extracted and purified
Define a recombinant DNA molecule
two or more different DNA molecules joined together
Summarize the process of cloning
(1) Insert purified DNA isolated via restriction digestion or PCR
(2) Plasmid (vector) cut open with a restriction digestion and insert digested with restriction enzymes to make matching ends, then ligase joins insert and plasmid backbone
(3) Transform ‘competent’ bacteria with ligation products
Describe the cloning rationale.
Which is true about reverse transcriptase?
Only found in bacteria
transcribes RNA into DNA
is not used in gene cloning
only found in the nucleus
none of the above
Always transcribes RNA into DNA
Describe reverse transcription (copying genes from mRNA).
Reverse transcription converts mRNA to cDNA (complementary DNA: double stranded DNA copy of an RNA), because it is easier to isolate genes of interest from mRNA than the genome. However, part of the sequence to ‘prime’ reaction must be known.
Describe polymerase chain reaction (PCR), an in vitro system to amplify DNA fragments.
‘Making’ DNA, or excising and amplifying DNA between two primers for purification.
Note that the primers require a known DNA sequence.
What is required for a PCR reaction?
Template DNA- from cells
Primers- synthesized in vitro
dNTPs- to make more DNA
Buffer- to make ideal environment for enzymes
DNA polymerase- to synthesize DNA
- must be resistant to high temperature
- Taq polymerase is a hotspring bacteria
How is DNA purified?
Agarose gel electrophoresis separates DNA fragments
- DNA is negatively charged: migrates towards the positive pole
- DNA fragments will separate from each other based on size (bp)
- Long DNA fragments migrate slower through agarose matrix
DNA is observed in gels using a fluorescent DNA intercalator (Ethidium Bromide, etc.)
DNA can be extracted and purified from bands on agarose gels
Define plasmid.
a genetic structure in a cell that can replicate independently of the chromosomes, typically a small circular DNA strand in the cytoplasm of a bacterium or protozoan. Plasmids are much used in the laboratory manipulation of genes.
Note that many carry antibiotic resistance genes, which can be used as selectable markers.
What are vectors?
Vectors are small, transferrable, and replicable DNA molecules that must be recognized and replicated in a host, such as plasmids (bacteria) or phage DNA (virus)
What is a plasmid?
bacterial DNA
passed between bacteria
double stranded
circular
all of the above
All of the above
What are the essential assets of a functional plasmid re-engineered for cloning?
Describe the process of cloning with λ phage.
Capacity for genome packaging into virus is 45Kb
The 15Kb central portion of the λ-phage genome with lysogenic genes, dispensible for replication and packaging, is removed for cloning.
That leaves a possible insert of up to 15Kb
Inserts assembled in vitro as concatemers (series of genomes linked by COS sites) before being spliced into 45Kb single genomes and packaged.
Packaged phage ready for infecting live bacterial host for propagation.
How does DNA fuse into a recombinant molecule?
Restriction Endonucleases/Enzymes are the original molecular tools that enabled Recombinant DNA technology.
Restriction endonucleases/enzymes make site-specific cuts in DNA, called restriction sites.
Today there are other alternative ways to clone DNA fragments
- via recombinases and PCR-based approaches
- restriction enzymes remain key and useful tools in molecular genetics labs
What are the two tyoes of restriction enzymes categorized by how they cut DNA?
blunt-end cutting enzymes and overhang cutting enzymes
What is the purpose of ‘overhangs’ in re-ligation of an inert into a vector with restriction enzymes?
‘Overhangs’ provide ends for re-ligation
Easy to stick molecules back together by facilitating ligation so the fragments stay associated
What is the origin of restriction enzymes?
Evolved as a bacterial mechanism to target and destroy infection phage DNA.
Restriction endonucleases protect bacteria from foreign DNA and bacteria protects it’s own genome against RE by methylation.
Define restriction enzymes.
an enzyme produced chiefly by certain bacteria, having the property of cleaving DNA molecules at or near a specific sequence of bases.
Restriction enzymes cleave DNA at specific nucleotide sequences. Restriction endonucleases cleave double-stranded DNA.
Describe the process of ‘competent’ bacteria being transformed with ligation products/
Vector enters bacteria via electroporation or heat shock that opens pores in the bacteria for the vector to be pulled within. Note this does have a low probability of success.
Once successful, bacteria is grown to amplify product and then the product is isolated.
How do I know that the recombinant vector I produce carry the right gene?
Two selection strategies in cloning:
(1) Selection for plasmid background
- ensure that the only surviving bacteria carries a plasmid
(2) Selection for presence of an insert
- identify among surviving clones the one with recombinant plasmids (with insert)
Describe the selection strategy for presence of the plasmid and why it has a low success rate.
Most bacteria do not pick up the plasmid
Successfully transformed bacteria have the plasmid- including ampicillin resistance gene.
Plate of transformed bacteria will be the plate that survives with an antibiotic (ampicillin)
