Lec 6-1 Flashcards
Recombinant DNA techniques
Gene cloning
Restriction enzymes
Visualizing DNA
Cloning genes
Using plasmid vectors (artificial DNA engineered to carry custom DNA sequences and genes)
Transformation of host cells with plasmids
Screening cells for recombinant plasmids
Gene cloning
Plasmids and bacterial transformation
We co-opt bacteria’s transformation ability to incorporate plasmid DNA for gene cloning
We can artificially make bacteria competent
Competent
Able to take up foreign walls by making the cell walls porous (can be done through heat shock)
Transformant
Bacterial cell that has taken in the plasmid
Replication can happen
Independent of bacterial cell genome using an origin of replication encoded in a DNA sequence in the plasmid
An ideal cloning vector has
An origin of replication
One or more selectable markers
Recognition sites for one or more restriction enzymes
Advantages of plasmids and bacterial transformation
Easy expansion- Bacterial cells can be quickly and cheaply expanded
Indefinite storage- Can be frozen and stored with little to no effect on bacterial cell health or degradation of DNA
Circular DNA is very stable
How do we obtain/purify only transformants
Through one or more selectable markers
Selectable markers
Genes that encode a marker, typically visual or growth selective
Allows only transformants to grow into colonies
A single colony is picked and cultured into large isogenic (same genotype) bacterial cultures
Plasmid DNA can be purified from these cultures
Restriction enzymes
DNA endonuclease enzymes
Discovered in bacteria
Defense mechanism against invading viruses
Functions as an innate immune system
Restriction enzyme have 3 ways cutting strands
5’ overhang
3’ overhang
blunt end
Restriction enzymes and DNA ligases
Two DNA sources cut with same enzyme and mixed
Overhang ends will hybridize due to the correct homology
Brightness of gel in ladders tells us
Size and how much is present in each ladder
Challenges of molecular genetics
DNA is extremely thin
Finding 1 gene specifically is like finding a needle in a haystack
Recombinant DNA capabilities
Locate a gene/DNA sequence
Remove/Copy DNA sequences
Visualize DNA
Store new DNA sequences
Recombinant DNA means
Combining DNA from different sources
Plasmids
Small circular double stranded DNA that can naturally exist in bacterial cells along side the bacterial genome
T4 DNA ligase is from
Bacteria
T4 DNA ligase
Covalently seals (ligates) nicks in sugar-phosphate bonds on two ends
Thus, creates stable double stranded piece of DNA
Restriction enzymes and DNA ligases allow us to
Cut, insert, and reorganize different DNA sources
Thus— recombinant DNA
Cloning vectors
Recognition sites for one or more restriction enzymes
Can have many different restriction sites
Restriction enzymes are named such that
the first portion in italics represents the species of bacteria they were discovered in
Visualizing DNA through gel electrophoresis
Gels are made that consist of a matrix with tiny holes
One side of the gel has small wells where DNA can be loaded
visualizing DNA through gel electrophoresis
Gels covered in ionic buffer and an electrical field is applied
Acids are negatively charged in this buffer
DNA molecules will slowly move toward the other end of the gel (positive end)
Gel electrophoresis
Larger DNA fragments move
Slower through the matrix, thus run slower than small DNA fragments
To visualize DNA in the gel
Gel electrophoresis
DNA is stained with something that binds nucleic acids, such as Ethidium Bromide
This can be imaged with a camera and an appropriate light source
Certain DNA bands can be cut out and purified
Polymerase chain reaction capabilities
Locate a gene/DNA sequence
Remove/Copy DNA sequence
Polymerase Chain Reaction (PCR)
Allows us to copy and then multiply a specific DNA sequence in an efficient matter
PCR is developed by
Kary Mullis
PCR is inspired by
How cells replicate DNA
