21 - Recombinant DNA Technology

Question 1

recombinant DNA

Answer 1

combined DNA of two different organisms

Question 2

transgenic/genetically modified organisms

Answer 2

organism containing recombinant DNA

Question 3

in vivo

Answer 3

transferring fragments to host cells using vectors

Question 4

in vitro

Answer 4

using polymerase chain reaction

Question 5

stages in process of making a protein using in vivo cloning

Answer 5

1. ISOLATION of DNA fragments containing gene for desired protein
2. INSERTION of DNA fragment into a vector
3. TRANSFORMATION - transfer of recombinant DNA into suitable host cell
4. IDENTIFICATION of host cells which have successfully taken up the gene
5. GRWOTH/CLONING of the population of successful host cells

Question 6

methods of producing DNA fragments (isolation)

Answer 6

conversion of mRNA to cDNA using reverse transcriptase
using restriction endonucleases to cut fragments containing the desired gene from DNA
creating the gene in a gene machine

Question 7

using reverse transcriptase to isolate a DNA fragment

Answer 7

mRNA which codes for specific protein is present
mRNA acts as template on which a single stranded complementary copy of cDNA is formed using reverse transcriptase
single stranded cDNA is isolated by hydrolysis of the mRNA with an enzyme
double stranded DNA is formed on the template of the cDNA using DNA polymerase
copy of desired gene is produced

Question 8

using restriction endonucleases to isolate a DNA fragment

Answer 8

each restriction endonuclease cuts a DNA double strand at a specific sequence of bases called a recognition sequence

Question 9

blunt ends

Answer 9

restriction endonuclease produces a straight cut

Question 10

sticky ends

Answer 10

restriction endonuclease produces staggered cut

each strand of DNA has several exposed unpaired bases

Question 11

gene machines

Answer 11

1. desired sequence of nucleotide bases for the gene is fed into the computer
2. sequence is checked for biosafety and biosecurity to ensure it meets international standards and ethical standards
3. computer designs a series of small overlapping single strands of nucleotides
   (OLIGONUCLEOTIDES) which can be assembled into the desired gene
4. each of the oligonucleotides is assembled by adding one nucleotide at a time in the required sequence
5. the oligonucleotides are joined together to make a gene (no introns)
6. gene is replicated using PCR

Question 12

promoter

Answer 12

region of DNA which RNA polymerase attaches to and starts transcription

Question 13

terminator

Answer 13

region of DNA which releases RNA polymerase and ends transcription

Question 14

insertion of DNA fragment into vector using restriction endonuclease

Answer 14

promoter and terminator regions added to the fragment
restriction endonuclease which was used to cut the DNA fragment is used to cut the plasmid
this ensures sticky ends of plasmid are complementary to sticky ends of vector
DNA fragments are mixed with the opened-up plasmids and become incorporated into the plasmids
the join is made permanent by DNA enzyme ligase

Question 15

transformation

Answer 15

reintroduction of plasmids containing recombinant DNA into bacterial cells
plasmids and bacterial cells mixed together in a medium containing calcium ions
ions make the bacterial membrane permeable
allows plasmids to pass through cell surface membrane into cytoplasm

Question 16

not all bacterial cells will possess vector with recombinant DNA after transformation:

Answer 16

only a few bacterial cells take up plasmids when they are mixed
some plasmids will have closed up without incorporating DNA fragment
sometimes DNA fragment ends join together to form its own plasmid

Question 17

method to identify which bacterial cells have taken up plasmids

Answer 17

all bacterial cells are grown on medium that contains antibiotic ampicillin
bacteria which have taken up gene will have acquired gene for ampicillin resistance
these bacteria are able to break down ampicillin and survive
bacterial cells which have not taken up the plasmids are not resistant to ampicillin and therefore die

Question 18

gene markers are easily identifiable

Answer 18

may be resistant to antibiotic
may make a fluorescent protein
may produce an enzyme whose action is easily identified

Question 19

antibiotic resistant marker genes

Answer 19

antibiotic resistance gene is cut in order to incorporate required gene
as the antibiotic resistance gene has been cut, it will no longer produce the enzyme which breaks down the antibiotic
therefore bacteria which have taken up desired gene are no longer resistant to that antibiotic
issue is that this process destroys all the cells which contain the required gene

Question 20

fluorescent marker genes

Answer 20

a gene which produces a green fluorescent protein (GFP) can be inserted into a plasmid
desired gene is transplanted into centre of the GFP gene
any bacterial cell which takes up the desired gene will not produce GFP

Question 21

enzyme marker genes

Answer 21

required gene is transplanted into the gene which codes for lactase production
if a plasmid containing desired gene is present in a bacterial cell, the bacteria will not produce lactase and so will not turn colourless substrate blue

Question 22

polymerase chain reaction PCR, requirements

Answer 22

DNA fragment to be copied
DNA polymerase
primers
DNA nucleotides
thermocycler

Question 23

primers

Answer 23

short sequence of nucleotide
has bases complementary to those at one end of each strand in the DNA fragment
provide the starting sequences for DNA polymerase to begin DNA copying
prevent separate strands from just rejoining

Question 24

thermocycler

Answer 24

computer controlled machine which varies temperature precisely over a period of time

Question 25

stages of PCR

Answer 25

1. separation of DNA strand - DNA fragments, primers and DNA polymerase placed in thermocycler vessel and heated to 95 C, strands in the DNA fragment separate
2. annealing of primers - mixture cooled to 55 C, primers anneal to their complementary bases at the end of the DNA fragments
3. synthesis of DNA - temperature increased to 72 C, optimum temperature for DNA polymerase to add complementary nucleotides along each of the separated DNA strands

Question 26

advantages of in vitro

Answer 26

extremely rapid
can replicate a minute amount of DNA fragments to produce large quantities of the fragment
doesn’t require living cells - no complex culturing techniques (which require time and effort) are needed

Question 27

advantages of in vivo

Answer 27

useful where we wish to introduce a gene into another organism
involves almost no risk of contamination - same restriction endonuclease used to open plasmid as to cut gene fragment (contaminant DNA will not be taken up by plasmid)
very accurate
cuts out specific genes - very precise
produces transformed bacteria - can be used to produce large quantities of gene products (eg. insulin)

Question 28

DNA probes

Answer 28

short single stranded length of DNA that has some sort of label attached to make it easily identifiable

radioactively labelled probes with isotope P-32
fluorescently labelled probes

Question 29

DNA hybridisation

Answer 29

when a section of DNA or RNA is combined with a single stranded section of DNA which has complementary bases

two strands are separated by heating - denaturation
when cooled, complementary bases on each strand anneal with each other to give the original double strand

Question 30

VNTRs

Answer 30

variable number tandem repeats
an organism’s genome contains many VNTRs
probability of two individuals having the same VNTRs is very low

Question 31

gel electrophoresis

Answer 31

1. DNA extracted from sample and its quantity is increased using PCR
2. restriction endonuclease cut the DNA into fragments
3. fragments separated using gel electrophoresis
4. gel is immersed in alkali to separate double strands into single strands
5. DNA probes are added to label fragments, these radioactive probes attach to specific fragments
6. X ray film is used, radiation from the probes exposes the film