recombinant DNA technology

Question 1

what is recombinant dna technology

Answer 1

involves transfer of fragments of DNA from one organsim/ species to another

Question 2

wht is the DNA of two or more organisms that has been combined called

Answer 2

recombinant DNA

Question 3

why is it possible

Answer 3

genetic code, transcription and translation machinary universal between all species,
trnsfered DNA fragemtns can be translated within cells of recipient organism (transgenic/GMO)

Question 4

stages of making a protein

Answer 4

isolation
insertion
transformation
identification
growth/cloning

Question 5

3 ways to form/ isolate DNA fragments

Answer 5

reverse transcriptase
restriction endonucleases
gene machine

Question 6

reverse transcriptase

Answer 6

protein producing cell with large amounts of mRNA selected
reverse transcriptase uses mRNA as template to produce cDNA
cDNA isolated by hydrolysis of mRNA by enzyme
double stranded DNA formed on the template cDNA using DNA polymerase
copy of desired gene formed

Question 7

does cDNA have introns

Answer 7

no, mRNA doesnt have introns

Question 8

where do restriction endonucleases cut

Answer 8

recognition sequence (specific base sequence)

Question 9

gene machine order

Answer 9

protein > amino acid order > mRNA codons > DNA

Question 10

gene machine

Answer 10

DNA sequence etered into computer
checked for biosecurtiy, biosafety and ethics
computer designs oligonucleotides
computer build up oligonucleotides by joining individual nucleotides together
oligonucleotides joined to make a gene
DNA fragments amplified ( PCR or in vitro)
to make double strands and then replicate gene
gne insterted into bacterial plasmid which can replicate and transfer gene
new gene checled for mistakes
bacteria cells contianing gene cloned

Question 11

PCR

Answer 11

double stranded DNA fragments heated to 95, H bonds between base pairs break producing two single DNA strands
cooled to 55, allow primers to anneal to DNA fragments
heated to72, opt temp for DNA polymerase
DNA polymerase attaches nucleotides together to from 2 new double stranded DNA fragments

Question 12

what goes into PCR

Answer 12

DNA fragments
DNA polymerase
primers
nucleotides
thermocyclers

Question 13

number of dna fragments produced by a given nuber of cycles=

Answer 13

2^x
x= number of cycles
(2 fragments produced pe crycle)

Question 14

in vivo- transformation

Answer 14

DNA cut using restriction endonuclraases to create sticky ends
promoter and terminatoer region added to allow transcripton
same restirction endonuclease is used to cut open the plasmid to allow complimentary sticky end
enzyme: DNA ligase used to incorporate DNA fragment into plasmid
recombinant DNA formed
recombinant plasmid transferred into bacteria via heat shock and Ca2+

Question 15

how can we identify which bacterial cells have taken up recombinant plasmids

Answer 15

marker genes

Question 16

why are marker genes useful

Answer 16

not all plasmids take up foreign DNA fragments
not all recombinant plasmids will be taken up by bacteria cells

Question 17

identify if plasmid was taken up

Answer 17

all bacterial cells grown on medium that contains antiobiotic ampicillin
bacterial cells that have taken up plasmid have acquired the gene for ampicillan resistance
these bacterial cells break down ampiciilain and survive
bacterial cells that have not taken up plasmids will not be reistant-die

Question 18

identify if recombiannt palsmids are taken up

Answer 18

insert DNA fragment into maker gene
if function is performed, recombinant DNA has not been taken up
if function is not performed, recombinant DNA has been taken up

Question 19

identify if plasmid was taken up

Answer 19

all bacterial cells grown on medium that contains antiobiotic ampicillin
bacterial cells that have taken up plasmid have acquired the gene for ampicillan resistance
these bacterial cells break down ampiciilain and survive
bacterial cells that have not taken up plasmids will not be reistant

Question 20

how to identify living colonies of bacteria containing required gene

Answer 20

replica plating

Question 21

gene therapy

Answer 21

mechanism by which genetic disorders can be cured or treated by masking thr effect of the fualty allele with the insertion of the functional allele