in vivo and in vitro cloning Flashcards
whats recombinant DNA
contains genes from two species
what are the three ways of producing DNA fragements
using reverse transcriptase
using restriction endonuclease
the gene machine
explain how reverse transcriptase produces DNA fragment
cell that readily produces the protein is selected
have large quantities of relevant mRNA
reverse transcriptase makes cDNA from RNA
complementary nucleotides pair with new cDNA strand and DNA polymerase joins new nucleotides
double strand has the gene for the selected protein
explain how restriction endonucleases form dna fragments
cuts a dna double strand at a specific sequence of bases - recognition sequence
cut DNA in staggered fashion known as sticky end
whats a sticky end
allows you to join two pieces of DNA cut by the same restriction endonuclease by complementary base pairing. the base sequences cut in this way are palindromic
how to produce dna fragment using gene machine
desired protein has specific base sequence put into the computer
sequence checked for biosafety
produces oligonucleotides which are joined together to make a gene
once fragment of dna produced theres two ways to clone it what are they
in vitro - using pcr
in vivo - using vector
describe full process of in vivo cloning
cut desired gene from other organism using restriction endonuclease or reverse transcriptase
use restriction endonuclease to produce complementary sticky ends
use same enzyme to cut plasmid
mix together and use DNA ligase to join wanted gene to plasmid or other gene
if a plasmid - return to cells and place in solution of Ca2+ which make the bacteria cell walls and membrane more permeable allowing plasmid to be taken up
how can you identify if a plasmid has been taken up by a bacteria cell
plasmids may contain gene that codes for fluorescence protein. lets them glow under UV light. allows quick identification of transformed bacteria under UV light
may contain antibiotic resistance genes. these act as a marker gene. allows detection of cells containing plasmid/DNA
whats a vector
carrier of DNA/gene
into cell or other organism
whats a DNA probe
single stranded dna
base sequence complementary to DNA/ gene that is being identified
radioactively/ fluorescently labelled so it can be detected
whats the polymerase chain reaction?
used to make copies of a gene or amplify small amounts of DNA
describe in vitro cloning
PCR:
heated to 95 degrees so H bonds between nucleotides so the strands separate
cooled to 52 degrees allowing primers to join to DNA. add nucleotides
re heated to 72 degrees so dna polymerase can add complementary nucleotides along each separate dna strand.
repeat cycle many times
what does a primer do
mark region of DNA to be copied
show enzyme where to start
enzyme needs starting strand onto which to attach nucleotides
