21 - Recombinant DNA Technology Flashcards
recombinant DNA
combined DNA of two different organisms
transgenic/genetically modified organisms
organism containing recombinant DNA
in vivo
transferring fragments to host cells using vectors
in vitro
using polymerase chain reaction
stages in process of making a protein using in vivo cloning
- ISOLATION of DNA fragments containing gene for desired protein
- INSERTION of DNA fragment into a vector
- TRANSFORMATION - transfer of recombinant DNA into suitable host cell
- IDENTIFICATION of host cells which have successfully taken up the gene
- GRWOTH/CLONING of the population of successful host cells
methods of producing DNA fragments (isolation)
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
using reverse transcriptase to isolate a DNA fragment
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
using restriction endonucleases to isolate a DNA fragment
each restriction endonuclease cuts a DNA double strand at a specific sequence of bases called a recognition sequence
blunt ends
restriction endonuclease produces a straight cut
sticky ends
restriction endonuclease produces staggered cut
each strand of DNA has several exposed unpaired bases
gene machines
- desired sequence of nucleotide bases for the gene is fed into the computer
- sequence is checked for biosafety and biosecurity to ensure it meets international standards and ethical standards
- computer designs a series of small overlapping single strands of nucleotides
(OLIGONUCLEOTIDES) which can be assembled into the desired gene - each of the oligonucleotides is assembled by adding one nucleotide at a time in the required sequence
- the oligonucleotides are joined together to make a gene (no introns)
- gene is replicated using PCR
promoter
region of DNA which RNA polymerase attaches to and starts transcription
terminator
region of DNA which releases RNA polymerase and ends transcription
insertion of DNA fragment into vector using restriction endonuclease
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
transformation
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
not all bacterial cells will possess vector with recombinant DNA after transformation:
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
method to identify which bacterial cells have taken up plasmids
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
gene markers are easily identifiable
may be resistant to antibiotic
may make a fluorescent protein
may produce an enzyme whose action is easily identified
antibiotic resistant marker genes
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
fluorescent marker genes
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
enzyme marker genes
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
polymerase chain reaction PCR, requirements
DNA fragment to be copied DNA polymerase primers DNA nucleotides thermocycler
primers
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
thermocycler
computer controlled machine which varies temperature precisely over a period of time
stages of PCR
- 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
- annealing of primers - mixture cooled to 55 C, primers anneal to their complementary bases at the end of the DNA fragments
- synthesis of DNA - temperature increased to 72 C, optimum temperature for DNA polymerase to add complementary nucleotides along each of the separated DNA strands
advantages of in vitro
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
advantages of in vivo
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)
DNA probes
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
DNA hybridisation
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
VNTRs
variable number tandem repeats
an organism’s genome contains many VNTRs
probability of two individuals having the same VNTRs is very low
gel electrophoresis
- DNA extracted from sample and its quantity is increased using PCR
- restriction endonuclease cut the DNA into fragments
- fragments separated using gel electrophoresis
- gel is immersed in alkali to separate double strands into single strands
- DNA probes are added to label fragments, these radioactive probes attach to specific fragments
- X ray film is used, radiation from the probes exposes the film
