Meeting 10 & 11

• 1) what's it's function?
• 2) where is it located?
• 3) what's its structure?

• can use THREE tools to figure out the answers to these questions:
• 1) the GENE that encodes the protein
• 2) a mutant cell line that LACKS the function of the protein
• 3) some of the actual protein so it can be studied

• -start with a mutant defective in some process of interest
• -isolate the affected gene
• -isolated gene can be manipulated to produce LOADS of the protein for experimentation

same essential steps; reverse the order

• -begin by isolating a protein of interest
• -find the corresponding gene (easy [ish] because we have most genome's sequenced)
• -gene can be altered and reinserted back into an organism to see how the mutation affects it

recessive = mostly inactivation of a gene; leads to a LOSS of funciton

dominant mutations lead oftentimes to a GAIN of function

ethylmethane sulfonate; agent commonly used to induce mutations in experimental organisms; does so by modifying G bases; leading to a mutation of G*C pairs to A*T pairs

THIS IS CALLED A POINT MUTATION DUH

no change in the amino acid sequence of a protein

point mutation that results in substitution of one amino acid for another

point mutation that results in a premature stop codon, aka in the middle of an amino acid sequence

point mutation that changes the reading frame of the gene

simpy any DNA molecule composed of sequences derived from different sources

restriction enzymes and DNA ligaases

endonucleases produced by bacteria that recognize 4-8 bp sequences (restriction sites) and cleave BOTH strands of DNA at these sites

• bacteria also produce a modification enzyme for EVERY restriction enzyme produced; these PROTECT a bacteria's OWN genome from cleavage by protecting or modifying at a potential cleavage site
• -specifically a METYL group is added within 1 or 2 bases of the restriction sequence/site

SmaI and AluI generate blunt end cuts

• ex. SmaI: CCC^GGG
• GGG^CCC

4⁸ base pairs (~65kb); the fragments that result from such cleavages are called restriction fragments

DNA ligase from bacteriophage T4 can ligate an two BLUNT DNA ends; but such ligation is inefficient and requires a greater concentration of both DNA and DNA ligase than does sticky end ligation

• 1) replication origin corresponding to the HOST
• 2) marker that permits selection
• 3) a drug resistant gene (so those vectors that take up the plasmid can be isolated)
• 4) region that can take up the DNA to be inserted (polylinker)

the 4th region necessary in a plasmid; it's a synthetically generated sequence that has ONE copy of different restriction sites NOT present elsewhere in the vector; when the vector is treated with one of these specific enzymes, it opens up only at one site (with stick ends) and if the DNA that's going to be inserted has also been treated with said enzyme the ends can be ligated together.

an entire genome is fragmented and individual fragments are cloned into a vector

• 1) mRNA is isolated from cells (easy to distinguish from other types of RNA because polyA tails hybridize to oligo-dT primers!)
• 2) reverse transcribed into cDNA (mRNA removed using alkali)
• 3) individual cDNAs are cloned into a vector

• 1) introns make the actual genes too long to be inserted into plasmid vectors
• 2) again, the presence of introns and intergenic regions make it difficult to identify the actual coding sequences (what's important)

this is solved by using cDNA libraries

the ability of complementary SINGLE-stranded DNA or RNA molecules to associate specifically with each other via base-pairing

20 bp; long enough to it's sequence to occur uniquely; occurs every 4²⁰ nucleotides; means it usually only occurs once in a genome

type of vector capable of replication in two different hosts

• contains 3 EXTRA elements in addition to all those found in the plasmid vector (so in total it has 6 important regions)
• 1) ARS: ORI for DNA in yeast
• 2) CEN: yeast centromere which allows for segregation of the plasmid during yeast cell division
• 3) URA3: gene that codes for an enzyme that makes uracil; selectable marker

polylinker, ORI (bacterial), amp^r selective gene, CEN, ARS (yeast ORI), and finally URA3 (yeast selective marker for uracil)

restoring wild-type function by introducing a functional copy of a gene into an organism in which that gene is mutated

incorporation of a ddNTP terminates strand elongation; instead of a 3' hydroxyl group there's just an H atom; cannot form a phosphodiester bond (aka dehydration reaction can't occur) with the next incoming nucleotide

blah blah blah

• 1) it's slow
• 2) constrained by the size of DNA fragment the plasmid is capable of holding
• 3) have to have convenient restriction enzyme sites

should be able to narrate that video

• 96---heat denaturing
• 50-60 --- primer annealing
• addition of Taq pol (can synthesize DNA even at high temperatures, aka at temperature where the two original strands or subsequently created strands won't reassociate)
• 72 --- complementary DNA strand synthesis

REPEAT

it's only after the 3rd cycle that the DNA sequence length of choice is actually present in the mixture