Gene technologies allow the study and alteration of gene function Flashcards
what is recombinant DNA?
transferred fragments of DNA from one organism, or species, into another
why can the transferred DNA be translated in the host cell?
- DNA is universal
2. transcription and translation are universal mechanisms
what are the methods for forming DNA fragments?
- conversion of mRNA to complementary DNA (cDNA) using reverse transcriptase
- using restriction endonucleases to cut a fragment containing the desired gene from the DNA
- creating the gene in a gene machine
describe how reverse transcriptase is used to form DNA fragments
- mRNA is isolated from a cell that readily synthesizes the protein coded for by the desired gene
- mix mRNA with DNA nucleotides and reverse transcriptase; RT uses the mRNA as a template to form a single strand of complementary DNA
- Add more DNA nucleotides and DNA polymerase forms the 2nd strand of DNA using cDNA as the template; forms double stranded DNA= desired gene
describe how restriction endonucleases are used to form DNA fragments
different restriction endonucleases cut DNA at specific sequence of bases- ‘recognition sites’
the shape of the recognition site is complementary to the active site
some restriction enzymes cut in a staggered fashion
this forms sticky ends
what are the 2 outcomes of using restriction endonucleases to form DNA fragments?
blunt ends and sticky ends
describe how the gene machine is used to form DNA fragments
- Examine the desired protein and identify the amino acid sequence
- mRNA codons are looked up and the complementary DNA triplets are worked out
- The DNA sequence of nucleotides bases is fed into a computer
- Checked for biosafety and biosecurity (the protein being produced is safe and ethical to produce)
- The computer designs small overlapping single strands of nucleotides called oligonucleotides which can be assembled into a gene
- The oligonucleotides are joined to make a gene🡪 it does not contain introns or non coding DNA
- The gene is amplified using PCR and then made into a double stranded gene
- The gene can be inserted in to a bacterial plasmid using sticky ends. It is now a vector which can be stored, cloned or transferred
what are the advantages of using reverse transcriptase to form DNA fragments?
- mRNA present in cell from actively transcribed genes
2. lots of mRNA to make cDNA
what are the advantages of using restriction endonucleases to form DNA fragments?
sticky ends on DNA fragments make it easier to insert into a vector to form recombinant DNA
what are the advantages of using the gene machine to form DNA fragments?
exact DNA fragment synthesized
what are the disadvantages of using reverse transcriptase to form DNA fragments?
lots of steps so very time consuming; more difficult
what are the disadvantages of using restriction endonucleases to form DNA fragments?
DNA fragment has introns
what are the disadvantages of the gene machine to form DNA fragments?
need to know the amino acid sequence
what are the 2 methods to amplify DNA fragments?
- in vivo; culture of transformed host cells
2. in vitro: PCR
describe the how enzymes can be used to insert a gene/DNA fragment into a plasmid (3 marks)
- Restriction endonuclease is used to cut the plasmid, the same restriction endonuclease is used to cut the DNA fragment; forming sticky ends
- so they have complementory sticky ends
- DNA ligase is used to join the 2 DNA strands to gether; by forming phosphodiester bonds
what must be done to the DNA fragments in a culture of transformed host cells to allow the DNA fragments to be amplified
- add promotor and terminator regions
2. allows gene to be transcribed and translated into a protein
what are methods of identifying if a host cell has taken up the recombinant DNA?
- antibiotic resistance markers
- fluorscent markers
- enzyme action markers
how does antibiotic resistance show host cells have taken up the recombinant DNA?
- antibiotic resistance acts as a marker
- bacteria cell contains 2(or more) antibiotic resistant genes
- one of these genes is disrupted when DNA fragment is inserted
- recombinant plasmid is then inserted into the bacteria cell
- bacteria cell is grown on agar; treated with one of the antibiotics
- cells that have taken up that plasmid will be resistant
- Replica plate grown; treated with the other antibiotic
- only those that have taken up the recombinant DNA will die out (as there is no longer any resistance)
how do fluorescent markers show host cells have taken up recombinant DNA?
- gene for fluorescence is inserted into a plasmid; acts as a marker (GFP)
- DNA fragment is inserted into the fluorescent gene (gene is disrupted, cannot be expressed)
- only those bacteria cells that DID take up the recombinant DNA will NOT glow
how does enzyme action show host cells have taken up recombinant DNA?
- enzyme action acts as a marker
- DNA fragment inserted into the gene for lactase
- lactase turns certain colorless solutions blue - insertion disrupts the lactase gene; not expressed
- colorless spots on agar show host cells have taken up recombinant DNA
why do not all bacteria cells take up the recombinant DNA?
- recombinant plasmid doesnt get inside cell; cell surface membrane not permiable enough
- plasmid rejoins
- DNA fragment rejoins; forms a plasmid
how are bacteria host cells transformed in order to be able to take up the recombinant DNA?
- placed in ice-cold calcium ion solution
- heat chocked
- allows cell surface membrane to become more perimiable
what are the 3 overall steps of PCR as a method to amplify specific DNA fragments?
- strand separation (95)
- annealing (55)
- DNA strand synthesis (72)
what is needed to carry out PCR as a methods of amplifying specific DNA fragments?
- thermocycler
- DNA fragment
- DNA polymerase (taq polymerase)
- DNA primers
- Nucleotides
describe the process of PCR as a method of amplifying specific DNA fragments
- at 95, seperate DNA strands by breaking weak hydrogen bonds between the bases
- At 55, allow primers to anneal to DNA fragment strand, forming hydrogen bonds
- primer is complemtary to template DNA
- DNA polymerase binds primer to start synthesis; adds nucleotides from the 3’ end to 5’
- 2 different primers required as DNA is aniparallel
- At 72, DNA nucelotides align next to complmentary exposed bases
- DNA polymerase joins adjacent nucleotides, forming phosphodiester bonds
why is taq polymerase used?
enzyme can withstand higher temperatures for PCR that human DNA polymerase cannot
what is the function of the primers in PCR?
- marks the region of DNA that needs to be replicated
2. enzymes needs a starting strand in order to attach nucleotides
why do 2 different primers have to be used in PCR
- DNA strands are anti-parallel
2. nucleotides will only bind from the 3’ end to the 5’ end
explain why each temperature is used at each stage of PCR?
- 95- breaks the h-bonds holding complementary DNA bases
- 55- allows H-bonds to re-form and allows the primers to attach
- 72- optimal temperature for taq polymerase to function
what are some advantages to using PCR as a method of amplifying DNA fragments?
- rapid process
- doesn’t require living cells
- useful when we want to introduce a gene into another organism
what are some disadvantages to using PCR as a method of amplifying DNA fragments?
- risk of contamination by unwanted DNA
- cannot cut out specific gene
- doesn’t produce transformed bacteria
