Recombinant DNA technology

Question 1

recombinant dna techonology

Answer 1

transferring DNA fragments from one organism to another

genetic code is universal so same codon/ triplet code codes for same amino acids

Question 2

reverse transcriptase

Answer 2

gaining fragments of DNA
conversion of mRNA to cDNA
mRNA form target cell is isolated and combined with reverse transcriptase
forms a single strand of complementary DNA (cDNA)
DNA polymerase joins nucleotides to produce double stranded cDNA
copy of gene is produced

Question 3

restriction endonucleases

Answer 3

enzymes that recognise pallondromic sequences and cut DNA here
RE have specific sequences of bases- recognition sequences which is complementary to their active site + they cut DNA here
can cut open plasmid (in vivo cloning)
cut between opposite bases for blunt ends
cut staggered to form sticky ends
sticky ends form complementary base sequences on other pieces of DNA that have been cut with same RE (when entering gene into another organism)

Question 4

gene machine

Answer 4

DNA can be synthesised from scratch
computers generate nucleotide sequence to produce gene
automated process assembles gene by adding singular nucleotides
gene doesnt have introns + is replicated using PCR

Question 5

amplifying DNA

Answer 5

gaining large quantities of DNA

Question 6

in vivo cloning

Answer 6

uses bacteria as they increase in number rapidly and are easy to culture
plasmid= type of vector
DNA fragment is obtained
promoter and terminator regions are added to ensure transcription occurs
plasmid DNA fragments cut open using same RE as for dna fragment
DNA fragment inserted into plasmid
sticky ends of plasmid complementary to sticky ends of dna fragments
DNA lisage joins sticky ends of dna to vector dna
plasmids are transported into host cells
bacteria in medium with calcium ions and is heat shocked
marker genes can be used to identify transformed cells

Question 7

not all bacteria contain required gene

Answer 7

only a few plasmids get taken up by the bacterial cells

some of DNA fragments join ends and form their own plasmid

Question 8

marker genes

Answer 8

second gene inserted into plasmid with DNA fragment
may be resistant to specific antibiotic
may make fluorescent protein that can be seen
might produce an enzyme that can be identified

Question 9

PCR equipment

Answer 9

dna fragment
dna polymerase
primers- short sequences of nucleotides
nucleotides
thermocycler- computer controlled machine that varies temperatures precisely

Question 10

in vitro cloning

Answer 10

separation-DNA heated to 95 C
DNA fragments open up, breaks h bonds, forms 2 single strands which act as templates
annealing- cooled to 55C
primers bind to complementary bases at end of DNA fragments
provide starting sequence for DNA poly which can only act on double strand
nucleotides attach by complementary base pairing
synthesis- heated to 72 C
optimum temp for DNA poly
DNA poly joins complementary nucleotides to separated DNA strands forming 2 new DNA fragments
cycle is repeated