8.4 Gene technologies Flashcards
What does recombinant DNA technology involve?
Involves the transfer of fragments of DNA from one organism to another. Since the genetic code is universal, as are transcription and translation mechanisms, the transferred DNA can be translated within cells of the recipient (transgenic) organism
Describe the process of recombinant DNA technology:
- Isolation of DNA fragments containing the desired gene
- Insertion of DNA fragment into vector
- Transformation (transfer of DNA into host organism)
- Identification of host cells that have successfully taken up the gene, using gene markers
- Growth/cloning to increase the number of host cells
What are the 3 different methods in which fragments of DNA can be produced?
- Conversion of mRNA to complementary DNA (cDNA), using reverse transcriptase
- Using restriction enzymes to cut a fragment containing the desired gene from DNA
- Creating the gene in a ‘gene machine’
Describe the process of conversion of mRNA to complementary DNA (cDNA), using reverse transcriptase:
- mRNA isolated from a cell that readily synthesises the protein coded for by the desired gene
- Mix mRNA with DNA nucleotides and reverse transcriptase –> reverse transcriptase uses mRNA as a template to synthesise a single strand of DNA
- DNA polymerase is used to build up the complementary nucleotides on the cDNA, forms second strand of DNA (double stranded)
What is the advantage of using mRNA to make DNA fragments?
- More relevant mRNA in cell than DNA easily extracted
- Introns removed by splicing (in eukaryotes) whereas DNA contains introns
- Bacteria can’t remove introns
Describe the process of using restriction enzymes to cut a fragment containing the desired gene from DNA:
- Some restriction endonucleases have a recognition site that produces a straight cut and therefore blunt ends (less control) : shape of recognition site complementary to active site
- Some restriction endonucleases cut in a staggered fashion = ‘sticky ends’ formed : only inserted in one way therefore more control
Describe the process of creating the gene in a ‘gene machine’
- Desired sequence of nucleotide bases in a gene is determined from the amino acid sequence of the protein produced, and put into a computer
- Sequence is checked for biosafety and biosecurity
- Computer designs a series of small, overlapping single strands of nucleotides, called oligonucleotides, which are synthesised and assembled into the gene
- PCR is used to replicate the desired gene (double stranded)
- Gene cloning can be used to amplify the gene
- Genes are checked for errors before being inserted into host organism
What is the difference between in vivo and in vitro?
In vivo - host cell using a vector
In vitro - PCR (amplification of specific DNA fragments)
What does a PCR (polymerase chain reaction) require?
- DNA fragment
- Taq polymerase
- Forward/reverse primers
- Nucleotides
Describe the first step of the PCR:
Strand separation (95°C)
- Causes the two strands of the DNA fragments to separate due to the breaking of the hydrogen bonds
Describe the second step of the PCR:
Primer annealing (55°C)
- Mixture cooled to 55°C causing the primers to join to their complementary bases at the end of the DNA fragment. Primers provide the starting sequences for DNA polymerase to begin DNA copying because DNA polymerase can only attach nucleotides to the end of an existing chain.
- Two different primers required
Describe the third step of the PCR:
Extension (72°C)
- Optimum temperature for DNA polymerase to make complementary copies of DNA
- Nucleotides align next to complementary exposed bases
- DNA polymerase joins adjacent nucleotides, forming phosphodiester bonds
Why do promotor and terminator regions need to be added?
Need to be added in order for the gene to be transcribed and then translated into a protein
What are promotor regions?
DNA sequences that tell RNA polymerase when to start producing mRNA
What are terminator regions?
Tell RNA polymerase when to stop transcription
How are restriction endonucleases and ligases used to insert fragments of DNA into vectors?
- Vector transports DNA into host cell eg/plasmids
- Vector DNA and DNA fragment cut using same restriction enzyme
- Vector DNA and DNA fragment have complementary sticky ends –> complementary base pair
- DNA ligase forms phosphodiester bond between adjacent nucleotides on sticky ends
Describe the transformation of host cells using vectors?
- Once the DNA has been incorporated into at least some of the plasmids, they must then be reintroduced into bacterial cells
- Involves the plasmids and bacterial cells being mixed together in a medium containing calcium ions
What are the conditions used for transformation?
Calcium ions and changes in temperature - makes the bacterial membrane permeable, allowing the plasmids to pass through the cell-surface membrane
How come not all the bacterial cells possess the DNA fragments with the desired gene for the desired proteins?
- Only a few bacterial cells take up the plasmids when the two are mixed together
- Some plasmids will have closed up again without incorporating the DNA fragments
- Sometimes the DNA fragment ends join together to form its own plasmid
