Chapter 21: Recombinant DNA technology Flashcards
what is meant by recombinant DNA technology?
The transfer of DNA fragments from one organism to another
Why does recombinant DNA technology
work?
- because the genetic code is universal,
- therefore transcription and
translation are also universal mechanisms
therefore all the same DNA base triplets code for same AA in amino
acid sequence across organisms
(indirect evidence of evolution)
what is the name of the organism that receives transferred DNA fragment?
GMO (genetically modified organism) or transgenic
What is recombinant DNA?
- DNA formed artificially by combining DNA from two different organisms
- resulting organism is GMO/transgenic
Briefly describe the stages involved in making a protein using DNA technology (in vivo)
- isolation of DNA fragments (that have gene for desired protein)
NB: Promoter and Terminator must be added! - insertion of DNA fragment into vector
- transformation - transfer of DNA into suitable host cells
- identification of host cells that have successfully been taken up by gene (using gene markers)
- growth/cloning of population of host cells
Name the three processes that can be used in creating DNA fragments
- reverse transcriptase
- restriction endonucleases
- gene machine
Summarise the process of using reverse
transcriptase to produce DNA fragments
- cells that produce protein of interest selected (bc they produce lots of mRNA for that protein)
- mixed w/ free nucleotides which match/bind to complementary base pairs (form H bonds), reverse transcriptase forms sugar-phosphate backbone of cDNA (intron-free)
- to make cDNA double-stranded, add more DNA nucleotides and DNA polymerase
Summarise the process of using restriction endonucleases (enzyme) to produce DNA fragments
- RE complementary to recognition sites/sequences (a DNA base sequence)
- RE cut double stranded DNA to create staggered ends which are palindromic (a staggered cut will create blunt ends)
- sticky ends (exposed bases) aligned next to complementary exposed DNA bases of organism they’re being inserted into - organisms DNA cut with same RE so complementary to each other
(method still contains introns)
Summarise the process of the ‘gene machine’ to produce DNA fragments
- examine protein of interest and find AA sequence
- then mRNA sequence – then DNA sequence
- DNA base sequence entered into computer and oligonucleotides produces
- oligonucleotides = small sections of overlapping single strands of nucleotides that make up gene – joined to create DNA for entire gene
- PCR used to amplify and make double strand, using sticky ends the gene can be inserted into plasmid DNA
In which two ways can we amplify DNA
fragments?
● In vitro / polymerase chain reaction (PCR)
● In vivo / using host cells
Describe the reaction mixture in the first stage of PCR
vessel in thermocycler of PCR
contains:
- the DNA fragment to be amplified
- primers that are complementary to the start of the fragment
- free nucleotides to match up to exposed bases
- DNA polymerase to create the new DNA
Summarise the process of amplifying
DNA fragments using PCR
- Heat DNA to break H bonds between bases, separates DNA strands (95°C)
- Cooled to allow primers to bind (55°C), H bonds form to hold primers in place, nucleotides attach; by complementary base pairing;
- Heated again for optimum temp for DNA polymerase tojoin nucleotides together (72°C)
- New DNA acts as template for next cycle
what is a DNA primer? And the role of a DNA primer?
primer = short, single-stranded DNA sequence
- provides starting sequences for DNA polymerases as they can only attach new DNA nucleotides to an existing strand of nucleotides
Name the three stages of PCR
- Deanturing (95°C)
- Annealing (55°C)
- Synthesis (72°C)
Summarise the process of inserting a
DNA fragment into a vector
- plasmid is used as the vector, and is cut using the same RE used to cut out DNA fragment (so ends are complementary)
- DNA ligase joins the fragment and plasmid together
state the role of DNA ligase
catalyses the (condensation) reaction between nucleotides to form phosphodiester bonds
summarise the process of inserting vector into host cell (i.e. cell transformation)
- vectors/host cells mixed in ice-cold solution, then ‘heat-shocked’ (large,rapid change in °C)
- mixed with Ca2+ ions which also increase permeability of cell membrane of host cell (for vector to be taken up)
state three issues that occur that prevent cell transformation
- recombinant plasmid doesn’t get inside host cell (despite increased permeability of cell membrane )
- plasmid re-joins before DNA fragment entered
- DNA fragment sticks to itself (creating small loop), instead of inserting into plasmid
why do promoters and terminators need to be added to extracted DNA fragment at the start?
promoter = binding site for RNA polymerase to enable transcription to occur terminator = end of gene, causes RNA polymerase to detach and stop transcription SO only one gene at a time is copied into mRNA
Summarise the process of identifying
transformed cells: Genes
marker genes e.g. gene coding for fluorescence can be inserted into vectors along with DNA
when cells grow, UV light can be used to identify which cells have taken up vector and which haven’t
what are marker genes?
- genes that can be used to identify which bacteria successfully took up the recombinant plasmid
- added to vector at same time DNA fragment is
e.g. Antibiotic resistance
Fluorescence
Enzyme markers
Summarise the process of identifying
transformed cells: fluorescent markers
- gene with coding for fluorescence can be inserted into vectors along with the DNA - when cells begin to grow, UV light can be used to identify which cells have taken up the vector and which haven’t
or DNA fragment is inserted into gene that codes for fluorescence, it disrupts gene, so when grown and UV light used, the transformed cell can be identified as the one that does not glow
If a gene for antibiotic resistance is disrupted, what will happen when the bacteria is incubated with that antibiotic?
It will not grow on that antibiotic
i.e. it will die/be killed
If a marker gene for antibiotic resistance is added and the cell is transformed, what will happen when the bacteria is incubated with that antibiotic ?
It will grow on that antibiotic
i.e. not be killed by antibiotic
Summarise the process of identifying
transformed cells: enzyme markers
- e.g. lactase turns substances colourless to blue
- gene for this enzyme inserted into plasmid, DNA fragment inserted into middle of this gene, disrupting it
- bacteria grown in agar plate with colourless substance
- colonies which can’t turn from colourless to blue contain recombinant plasmid
What is the next step after successfully transformed cells have been identified?
- fermenter used to grow multiple copies of host cell with recombinant plasmid
- large, cloned population of host cell can then produce the protein coded for by inserted DNA fragment
What must the vector contain for the host cells to produce the protein coded by the DNA fragment?
Promoter and terminator regions
NB: These may already be in the vector DNA
State the advantages and disadvantages of using reverse transcriptase to extract DNA fragment
advantages:
lots of mRNA (from cell that produces proteins)
disadvantages:
more time-consuming and difficult (i.e. there are more steps)
