Recombinant DNA Technology

Question 1

recombinant

Answer 1

transfer of fragments from one organism to another
genetic code is universal + so are transcription and translation methods
fragments of dna- made by separating/ breaking dna strands into pieces
fragments made by reverse transcriptase, restriction endonuclease and gene machine

Question 2

reverse transcriptase

Answer 2

conversion of mRNA to cDNA
-mRNA is isolated and combined with reverse transcriptase
-forms a single strand of complementary DNA (cDNA)
-DNA polymerase joins nucleotides to produce double stranded comp DNA
-codes for specific gene
gene can be inserted into vector

Question 3

RT strengths

Answer 3

easy to find genes as specialised cells contain lots of specific mRNA
mRNA doesn’t have introns

Question 4

restriction endonuclease

Answer 4

enzymes that recognise palindromic sequences and cut DNA
different RE hydrolyse different specific sequences
RE have specific sequence of bases
blunt end- cut between two opposite bases
sticky ends- cut staggered and each dna strand has bases exposed

Question 5

RE inserting genes

Answer 5

sticky ends make it easy to insert desired gene
can form complementary base sequences on other pieces of DNA that have been cut with same RE
DNA ligase used when inserting a vector

Question 6

gene machine

Answer 6

dna can be synthesised from scratch
use computers to generate nucleotide sequence to produce gene
automated process assemble genes by adding singular nucleotides
gene doesn’t have introns and is amplified by PCR

Question 7

amplifying dna

Answer 7

once fragments of dna have been made
they can be amplified to gain large quantities of dna
in vivo cloning

Question 8

in vivo cloning- method

Answer 8

use bacteria as they increase in number rapidly and are easy to culture

• DNA fragment is isolated
• promoter and terminator regions are added to ensure replication
• vector DNA cut open using same restriction endonuclease
• DNA fragment inserted into vector
• sticky end of vector comp to sticky end of DNA fragments
• DNA lisage joins sticky ends of DNA of vector DNA
• add to bacteria

Question 9

transformation into bacteria

Answer 9

bacteria in medium- consists of calcium ions and is ice cold
plasmids are added and mixture is heat shocked
-bacteria will replicate with plasmid
-marker genes can be used to identify transformed cells eg cells with dna fragment
marker may be fluorescent
if gene is for antibiotic resistance, bacteria who take it up will be killed by the antibiotic

Question 10

in vitro cloning

Answer 10

PCR- polymerase chain reaction

need- DNA fragment, DNA polymerase, primers (short sequence of nucleotides), DNA nucleotides, thermocycler

Question 11

PCR method

Answer 11

cycle- SAS
separation- heated to 95C
DNA fragments open up, breaks hydrogen bonds, forms 2 single strands which act as templates

annealing- cooled to 55C
primers bind to comp bases at end of DNA fragment
primers provide starting sequence for dna poly and nucleotides to attach

synthesis- heated to 72C
optimum temp for dna poly
dna poly joins complementary nucleotides
cycle is repeated

Question 12

DNA replication stops in PCR

Answer 12

all of the primers have been used up

Question 13

transgenic organism

Answer 13

organism that contains a gene from another organism

they will produce the protein encoded by the gene

Question 14

gene therapy

Answer 14

somatic cell therapy- copies of a functional gene are inserted into body cells of a patient

germ cell therapy- copies of a functional gene are inserted into gametes

Question 15

GM crops

Answer 15

in order to meet global demand for food
resistant to herbicides- increase yield
resistant to pests- increase yield
enriched in vitamins- increases nutritional value
-organisms with desired characteristics are produced more quickly
-all organisms will have desired characteristic

Question 16

social and ethical implication of GM crops

Answer 16

lack of long term research of effects on human health
dont know if crops are safe for human consumption and for organisms around them
reduction in biodiversity

Question 17

dna ligase

Answer 17

joins sticky ends of dna fragment and plasmid vector

forms phosphodiester bonds

Question 18

how GM reduces expression of a gene

Answer 18

new RNA inserted will bind to normal mRNA
as it is complementary
ribosomes can’t bind
less transcription of specific gene
less translation of protein coded for by gene
less of process can occur

Question 19

not all bacteria contain required gene

Answer 19

only a few plasmids get taken up by the bacterial cells

some of DNA fragments join ends and form their own plasmid