Genetic Technologies Flashcards
define recombinant DNA technology
the transfer of DNA fragments from one organism to another
-first step is to produce/ isolate the fragments of DNA to be recombined with another piece of DNA
3 methods used to create fragments of DNA
-using reverse transcriptases to make DNA from mRNA
-use restriction enzymes to cut out a section of DNA from the genome
-use a gene machine to build a section of DNA using nucleotides.
process of using reverse transcriptase to produce DNA fragments
-a cell that naturally produces the protein made by target gene is selected
-these cells will contain lots of mRNA for the protein, which is complimentary to the target gene
-the reverse transcriptase enzyme joins the free DNA nucleotides with complementary bases to the mRNA sequence to make DNA.
-single stranded DNA is produced-> cDNA (complementary DNA as it matched the mRNA)
advantage of producing DNA fragments using reverse transcriptase
the cDNA is intron free- only contains the coding regions as it is based on the mRNA template strand
process of using restriction enzymes to produce DNA fragments
-restriction endonuleases cut DNA at specific sequences.
-naturally occur in bacteria as a defense mechanism
-each enzyme cuts DNA at a specific recognition sequence, complementary to their active site
-restriction enzymes form sticky ends (pieces of DNA that have exposed nucleotides on a single strand at each end)
-allows different fragments to be joined or annealed together, joining complementary base pairs -when cut with the same restricive endonuclease
-the restriction sequence is palindromic as they are complementary to each other
two ways DNA fragments can be amplified
-in vitro cloning- polymer chain reaction (PCR)
-in vivo cloning- using host cells
how is DNA fragment modified before inserting into a vector
-desired gene is isolated using restriction endonuclease enzymes- cut at staggered point to produce sticky ends
-promoter region and terminator regions are added to the gene to make sure the gene can correctly transcribe once in the host
->promoter: tells RNA polymerase here to start transcribing mRNA
->terminator: tells RNA polymerase when to stop
process of inserting DNA fragment into a vector
-the plasmid is cut using the same restriction endonuclease enzyme to create same sticky ends on both DNA fragment and plasmid.
-DNA fragment sticky ends have the complementary base pairs as on the sticky ends of plasmid
-DNA fragment and cut plasmid are combined together using ligase enzymes (anneals them)
-ligase enzymes catalyse condensation reactions to form phosphodiester bonds between the nucleotides.
what is a vector
something that can carry the isolated DNA fragment into the host cell e.g. plasmids, viruses and certain bacteria
process of inserting vector into host cell
-known as cell transformation
-cell membrane of host cell (usually bacteria) must be more permeable
-to increase permeability, host cells are mixed with Ca2+ and heat shocked (sudden increase in temperature)
-this enable vector to enter host cell’s cytoplasm
process of identifying transformed cells
marker genes are used to identify which bacteria succcessfully took up recombinant plasmid-they are inserted into vectors the same time the DNA is, 3 different marker genes used are:
-antibiotic resistance genes-> colonies of bacteria are grown on an agar plates that contains the antibiotic. only transformed bacteria with the resistance gene will survive + can then be selected
-genes coding for fluorescent proteins-> after they have been left to grow, a UV light can be used to identify colonies which will glow.-must be the ones with the target gene so can then be selected
-enzyme marker genes-> enzyme lactase can turn a certain substance blue from colourless, gene coding for lactase is inserted into plasmid. The DNA fragment is inserted into the middle of this gene to disrupt it, bacteria are then grown on agar plate containing colourless substance. Plasmids that cannot turn the substance blue must contain the recombinant plasmid.
reasons why host cell may not successfully take up a recombinant plasmid
-recombinant plasmid may not get inside the cell
-the plasmid re-joins to itself before DNA fragment enters
-DNA fragment sticks to itself, rather than inserting to plasmid
how is the host cell grown
fermenter is used to grow multiple copies of the host cell which have been identified with recombinant plasmid.
-this large cloned population can then produce the protein coded for by the inserted DNA fragment e.g. bacteria producing insulin from inserted insulin gene.
stages of in-vivo cloning
-desired gene is isolated using restriction endonuclease enzymes
-promoter regions and terminator regions are added
-gene is inserted into a vector-usually a plasmid
-transformation of host cell with recombinant plasmid
-identify transformed cells
-grow the host cell (clone/make copies of the gene)
in vitro cloning
uses the polymerase chain reaction (PCR) to make millions of copies of a fragment of DNA using an automated machine.
-fragments of DNA undergo a series of heating and cooling using a thermocycler to replicate them many times in a similiar way to DNA replication in nucleus
