biotechnology

how elisa works

antigen binds to wells of a microplate → sample added → antibody binds to antigen → enzyme-linked antibody added → substrate reaction → signal measured & concentration of target molecule is determined

gel electrophoresis

separates fragments based on their length

gel electrophoresis works by loading a mixture of fragments at one end of the gel (which is agarose; polyacrylamide) which

contains a microscopic network of pores then a voltage is applied across the gel → the fragments migrate toward the positive electrode because DNA is negatively charged, shorter fragments are closer to the positive electrode (farther from the starting well)

to see the results of gel electrophoresis a

stain must be applied

southern blotting

DNA

northern blotting

RNA

western blotting

protein

cloning of genes: DNA is separated using restriction nucleases then

the enzyme DNA ligase is utilized to join together DNA fragments to make recombinant DNA

cloning genes: DNA can be ligated into a bacterial plasmid (circular DNA molecule), which serves as a vector to

maintain the foreign DNA inside the bacterium → the bacterium replicates the plasmid DNA as it reproduces

cloning a mammal: isolate a somatic cell from one individual → isolate an egg from another individual of the same species →

remove nucleus from egg → inject somatic nucleus into empty egg →grow fused cell in vitro to produce early embryo → implant embryo in surrogate mother → clone of somatic cell donor is born

PCR: heat is utilized to separate double-stranded DNA, primers are

hybridized to complementary sequences in the 2 strands, DNA polymerase & nucleotides are added

RT-PCR: RNA is used as a template to synthesize complementary DNA (cDNA),

the enzyme reverse transcriptase (a type of DNA polymerase) adds DNA nucleotides complementary to the RNA nucleotides

knockout mice are used to determine the role of a specific gene by

a targeted disruption of the gene, the effects of the disruption are then observed in the mouse

examples of when a knockout mouse was used

p53; Dystrophin

knockout mouse is useful when

trying to find out how gene mutations affect organisms

CRISPR occurs

naturally in bacteria as a defense mechanism; one DNA sequence is repeated several times with unique sequences in between the repeats

CRISPR in bacteria: part of the bacteria’s immune system which

keeps bits of dangerous viruses around so it can recognize & defend against the virus the next time it attacks

Cas is

the second part of the defense mechanism, the proteins can precisely cut the DNA of invading viruses

CRISPR-Cas works by

Cas9 (enzyme) snips DNA, CRISPR collects DNA sequences that tells Cas9 exactly where to cut; all you have to do is feed the correct sequence to Cas9

CRISPR-Cas: after feeding Cas9 the sequence,

you can cut and paste bits of DNA sequence into the genome wherever you want