chapter 21 Flashcards
(33 cards)
recombinant DNA technology
combining different organisms DNA to enable scientists to modify and alter DNA to better industrial processes and medical treatments.
creating DNA fragments using reverse transcription
uses reverse transcriptase enzmye
occurs naturally in viruses e.g. HIV
1) reverse transcriptase enzyme converts RNA into DNA
2) select a cell with the desired protein
3) this cell will contain lots of mRNA copies for the protein.
4) reverse transcriptase enzyme joins DNA nucleotides with complimentary bases to mRNA sequence
5) single stranded DNA fragment produced
6) DNA fragment made double stranded by adding DNA polymerase enzyme
a cell only has two copies of each gene so difficult to access.
what is the advantage of using reverse transcription?
cDNA/single stranded DNA has no intorns as made from mRNA sequence
restriction endonucleases
cut DNA at palindromic sites called restriction sites
enzymes which cut up the DNA
occur naturally in bacteria as defence mechanism
different restriction endonuclease enzymes which have active ssites complimentary to different DNA base sequences
this is scalled the recognition sequence
enzymes cut DNA at specific location
some enzzzymes cut double stranded DNA at the same point
this creates blunt ends
some enzymes create exposed DNA bases and staggered ends
these staggered ends are palindromic- read the same forwards and backwards
referred to as stick ends as they can join DNA with complimentary base pairs.
gene machine
creating DNA fragments in the lba using a computerised machine.
examine protein of interest to identify amino acid sequence so the DNA and mRNA sequence can be identified
DNA is put into computer to check biosafety and biosecurity- to check that the DNA being produced is safe and ethical
computer can then make small sections of single stranded overlapping nucleotides that make up the gene- called oligonucleotides.
oligonucleotides are then joined together to create DNA for gene.
this method is
quick
accurate
produces intron free DNA
which can then be transcribed by prokaryotes.
pros and cons of using reverse transcription
pros- mRNA in cell actively transcribed from genes so lots of mRNA to make single stranded DNA
cons- many steps so more time consuming
technically more difficult.
pros and cons of using restriction endonucleases
pros- sticky ends of DNA fragments make it more easy to insert to create recombinant DNA
cons- still contains introns
pros and cons of using gene machine
pros- can design exact DNA fragments with labels, sticky ends and preferntial codons
cons- need to know amino acid/base sequence.
how are the DNA fragments modified and why?
DNA fragments need to be modified to ensure transcription of genes occurs
promoter region added to start of DNA fragment
promoter is a sequence of bases which RNA polymerase binds to, to initiate transcription.
terminator
added at the end of the gene
causes RNA polymerase enzyme to dettach and stops transcription.
so only one gene is transcribed at a time.
vectors
carry isolated DNA into host cell
most common is plasmids
-circular DNA
-separate from bacterial genome
-only a few genes.
inserting the DNA into the vector
plasmid cut open using restriction endonucleases to create sticky ends
so sticky ends of DNA fragments are complimentary to sticky ends of plasmids.
DNA ligase enzyme
cut plasmids and DNA fragments are joined together by DNA ligase enzymes
catalyses formation of phosphodiester bonds between nucleotides.
transformation
for the vector/plasmid with recombinant DNA to be inserted into the host cell, the host cell membrane’s permeability needs to be increased
-this is done by mixing it with calcium ions and heat shocking it/suddenly increasing temperature.
so now the vector can enter the host cell’s cytoplasm.
amplifying DNA fragments
once the DNA fragments have been isolated, they need to be cloned to create large quantiies
can be done in vivo or in vitro.
DNA can be amplified in vitro using PCR/polymerase chain reaction
this is done using an automated machine.
equipment used in PCR
-DNA polymerase enzyme- taq polymerase- found in bacteria in hot springs- optimum temperature is much higher/72 degrees/does not denature at high tempoeratures
-thermocyle- machine
-DNA nucleotides
-primer- short sequence of DNA complimentary to start of DNA sample.
3 stages of the PCR
denaturation
annealing
synthesis
PCR
DNA is heated to 95 degrees to break hydrogen bonds between complimentary bases- separating two strands- denaturation
DNA is cooled to 55 degrees so the primer can attach- annealing
snyhesis- DNA polymerase attaches complimentary free nucleotides and makes a new strand to align next to each template
DNA is heated to 72 degrees as this is optimum temperature for taq dna polymerase.
advantages of PCR
automated- so more efficient
rapid- 100B copies of DNA can be made within a few hours
doesn’t require living cells- less complex technology needed and rapid.
what are DNA probes?
what are they used for?
DNA probes are short single stranded pieces of DNA with labelled radioactivity or flurorescene.
used to locate specific allele on a gene
screen patients for drug response, heritable conditions and health risks
how is the DNA probe designed?
how is the patient’s DNA altered?
The DNA probe is designed with a complimentary base sequence to the allele being screened for.
The patient’s DNA is treated to make it single stranded and then it is mixed with the DNA probe.
denaturation and annealing
the patient’s DNA sample is heated to break the hydrogen bonds between complimentary bases and make it single stranded- denaturation
single stranded patient DNA is added to DNA probe
some of the patient’s DNA will anneal to the DNA but some single stranded DNA will anneal to the DNA probe.
how a specific allele is located
to locate a specific allele you need to know the DNA base sequence to create the DNA probe
you find the DNA sequence using DNA sequencing methods e.g. the sanger method
single stranded DNA fragment created using gene machine.
label is added e.g. radioactive nucleotide containing P32 isotope is added/ fluorescent label which emits light under UV light.
once hybridisation has occured, DNA is washed to remove any unbound DNA probe.
the presence of radioactive isotopes/fluroscence indicates that the allele you are testing for is present within the patient’s sample.
genetic screening
DNA probes can be used to screen for genetic disorders or cancer causing oncogenes
you can screen for multiple genetic disorders at the same time by using an array which has multiple DNA probes attached.
