Designer Genes: Biotech

PCR, molecular cloning, gel electrophoresis, error prone PCR, reverse transcription, Sanger sequencing, illumina sequencing, nanopore sequencing

PCR stands for

polymerase chain reaction

end product of pcr

millions to billion of specific DNA segment copies

PCR Steps:

denaturization, annealing, extension

PCR: Denaturization

DNA split into 2 strand via heat (hydrogen bonds)

PCR: Annealing

DNA amplified via primer, temp to high → primers don’t bind, temp to low → primers bind wrong, taq polymerase enzyme copies

PCR: Extension

DNA copied

reverse transcription

converting RNA to cDNA

molecular cloning, example → (recombinant plasmid)

insert a gene of interest into an organism (recombinant plasmid)

MC: Step 1

isolate gene of interest and amiply via PCR

vector

container like a plasmid that holds DNA

MC: Step 2 → prepare a vector

prepare empty delivery vehicle (vector), use restriction enzymes to open vector for DNA

MC: Step 3 → Recombination

glue in new gene into vehicle via dna ligase and shut it in via phosphodiester backbone/gibson/golden gate assemblies

MC: step 4 → Transformation

calium chloride neutralizes charges and sudden hea shock allows for plasmids to enter membrane

MC: Step 5 → Selectrion & Screening

only cell that took up plasmid grow on agar plates

gel electrophoresis

separtes DNA based on size and determines types of DNA

DNA sequencing objective

determine order of nucleotides in DNA strand

dna sequencing types

sanger, illumina, nanopore

knockout

permanently destroys gene function

knockdown

temporarily turns down or silence gene expression

complementation

adds a healthy copy of the gene back into a mutant cell to see if the missing trait is restored, proving the og mutation was the cause