Recombinant DNA and Genome Editing

Molecular cloning - Technique in molecular biology where a DNA of interest is cloned using living cells from things like yeast Organism cloning - cloning animals

• Select a plasmid, one that has the selectable marker • Isolate DNA seq of interest (create and amplify cDNA), 2 main ways to do this, PCR or cut and paste • Create recombinant DNA (with restriction enzymes), ligate the DNA seq into plasmid • Introduce recomb DNA to bact or other host, depends on application • Screen the bact, check that the bact you have does indeed have the recomb DNA • Isolate the recomb DNA for further verification

cloning vector - plasmid without the DNA of interest Recombinant DNA (expression vector)- the plasmid with the DNA of interest

cDNA - DNA but it does not contain any intronic sequences DNA has a lot of introns, things that get taken out and are not important. cDNA is not naturally occuring, but it is more efficient to transcribe it than DNA

• take out the mRNA form the RNA by getting a primer that attaches to the AAA tail, only mRNA has it. • Use reverse transcriptase to make DNA from the mRNA and the primer cDNA is shorter than the original DNA • Use PCR, amplify to make many more copies of cDNA do around 40 cycles, not all of the copies will be good • if the cDNA does not contain the restriction site make sure the primers in PCR can add it.

when you pick a primer, have it overshoot the edge of the cDNA. The overshoot will contain the restriction site when the cDNA is amplified from the primer it will contain the restriction site sequence for Kaiso they used ECO RI and Sac 1

multiple cloning (restriction site) - Where the dna of interest goes, must have dna seq that is recognized by multi restriction enz promoter sequence - initiates transcirption, controls its rate origin of replication - where replication starts, required for replication antibiotic resistance gene - for bacterial selection selectable marker - for mammalian selection

bacteria - the plasmid contains a resitance gene, if the bact does not have the plasmid then it will die mammalian selection uses other selection markers but is simiilar in principle ex neomycin

Also take a liposomes force plasmids into liposomes drop the liposomes into mammalian cells liposomes will fuse with membrane then some of the cells take the plasmid in.

Choose a site that is present in the plasmid but absent in the DNA seq of interest This would cause the enzyme to cut in the middle of the DNA of interest

This is so that the cut ends do not fuse with each other. the cDNA and the plasmid will be cut by the same 2 restriction enzymes so only the cDNA will fit with the cut plasmid ends

You can get the DNA seq from biopsy and isolate from PCR Get the cells from human tissue culture If there is already another expression vector that has Kaiso then you cat take it out of there

heat shock it to make holes in the membrane

Can also be used to make antibodies, Recombinant vaccines - take parts of the virus that is recognized by the immune syst and create vaccines from it. So you don’t use the whole virus

using technology that has been cloned CRISPR, to alter the organisms genome Can be used to correct mutations insert DNA sequences Remove sequences Like when you want to tag a protein to be more visible, easier to track within the cell

PCR - polymerase chain reaction Has 3 steps Denaturation annealing elongation Used to amplify the cDNA in molecular cloning and add the restriction site

Depends on whether you insert the expression vector in bacteria or mammal Bacterial selection - antibiotic resistance gene Mammalian selection -neomycin

heat shock bacteria to get the expression vector in use liposomes to get the expression vector into mammalian cell hope it gets incorperated, screen later

Single stranded - one of the two strands is damaged Base excision repair - enzymes come in and cut the damaged base out, the DNA repair machinery will read the intact strand and fill in the gaps Nucleotide excision repair - the damage changes the helical shape of the DNA, requires multiple nucleotides to leave Both of them use the intact template strandto fix the DNA

Double stranded breaks - both strands are broken Homologous recombination - happens in S phase or later (this is when the cells duplicate the genome, instead of 2 copies there are 4 copies of the gene, the cell will use the homologous chromosome that was duplicated during S phase as a template) Non homologous end joining (NHEJ) - Happens outside of S phase, in G1. This happens at the G1 check point. The cell will just glue the broken ends together. This will cause a deletion. The protein will no longer function as well because some of the DNA is gone. This can be helpful in gene editing, CRISPR can get rid of some DNA

stands for clustered regularly interspaced short palindromic repeats. endogenous immuen defence mech that is used by bacteria. can be used by Humans for gene editing

• CAs1, Cas2 natural to bacteria, approaches the viral genome, cleaves small piece of bacterial genome • Cleaved thing now called protospacer • Bacteria integrates the protospacer into its own genome, • After integrated its known as a spacer • This makes up the CRISPR array • CRISPR array gets transribed as RNA called pre-crRNA • Cas9 processes pre-crRNA into crRNA • If the bacteria is infected by the same bacteriophage again then it can recognize its RNA from the crRNA • Cas9 guides the crRNA to the identical sequence in the pphage genome • This signals cas9 to cleave the phage genome This stops subsequent infections

• a. researchers give synth sgRNA to Cas9, directing it to DNA of interest b. Cas9 is limited by PAM location c. Cas9/sgRNA bind to PAM d. perfect base pairing between sgRNA and DNA • causes Cas9 to do dbl strand cleave in targeted site • NHEJ is activated and can create deletion or insertion

the sickle cell disease It comes from adult hemoglobin babies have fetal hemogrlobin and do not get sickle cell disease. people usually stop making fetal hemoglobin when they get older use CRIPSR and Cas9 to delete the gene that makes the protein that promotes adult beta globin, Use non homologous eng joing (NHEJ) to recombine the ends in S phase Person will only make fetal hemoglobin