What is recombinant DNA?
the transfer of DNA from one organism to another producing combined DNA
Why is it possible for DNA from different species to be transferred between one another?
because the genetic code is universal (the same in all organisms) and transcription/translation is the same in all organisms so the proteins coded for are made in the same way
What are the stages in making a protein using DNA technology?
- isolation of DNA fragments that code for the desired protein
- insertion of the DNA into a vector
- transformation - transfer of DNA into suitable host cells
- identification that the gene has been taken up using gene markers
- growth/cloning of the host cell
What are the 3 methods to isolate a fragment of DNA?
- conversion of mRNA to cDNA using reverse transcriptase
- using restriction endonucleases to cut fragments of the desired gene out of DNA
- creating the gene in a gene machine
How is cDNA produced using reverse transcriptase?
- a cell that readily produces the desired protein is selected eg. beta cells producing insulin
- these cells contain mRNA which codes for the production of insulin
- mRNA acts as a template strand on which a single-stranded complementary DNA nucleotides join, forming cDNA using reverse transcriptase
- cDNA is isolated from mRNA, and double stranded DNA is formed on the template of cDNA using DNA polymerase
- this double stranded DNA is the required gene
How are restriction endonucleases used to isolate DNA fragments?
- restriction endonucleases cut double stranded DNA at a sequence of bases specific to the enzyme called the recognition site
- this leaves exposed unpaired bases called sticky ends which can bond to other complementary sticky ends
How can gene machines be used to isolate genes?
- the desired sequence of bases can be determined by the amino acid sequence of the protein, as from this you can determine the mRNA codons and complementary DNA triplets
- desired nucleotide base sequence is fed into a computer
- sequence is checked for biosafety and biosecurity
- the computer designs a series of small, overlapping, single strands of nucleotides called oligonucleotides which can be assembled into the desired gene
- the gene has no introns or non-coding regions
- PCR is then used to replicate the gene
What is the difference between in vitro and in vivo gene cloning?
in vitro = using PCR
in vivo = transferring fragments to a host cell using a vector
What is the importance of sticky ends?
- if the same restriction endonuclease is used to cut DNA, then all of the fragments produced will have ends that are complementary to one another
- once the complementary bases of the 2 sticky ends are paired, DNA ligase is used to bind together the 2 sections of DNA using phosphodiester bonds
- this allows the DNA of one organism to be combined with DNA from another organism
What is the process of the polymerase chain reaction?
- separation of the DNA strand - DNA fragments, primers and DNA polymerase are places into the thermocycler and the temperature is increased to 95 degrees, causing hydrogen bonds to break and DNA to separate
- addition (annealing) of primers - mixture is cooled to 55 degrees causing primers to anneal to their complementary bases at the ends of the DNA fragments - this provides the starting sequence for thermostable DNA polymerase
- synthesis of DNA - temperature is increased to the optimum for DNA polymerase to add complementary nucleotides along the separated DNA strands
How is DNA prepared for insertion into a vector?
promoter and terminator regions are added to DNA - this ensures that transcription will only occur on the target section of the gene
How is DNA inserted into a vector?
- restriction endonucleases are used to cut a specific region of the gene on a plasmid (usually at an antibiotic resistant gene)
- this is the same enzyme that was used to cut the DNA fragment, which ensures that the sticky ends of the plasmid are complementary to the sticky ends of DNA so they join
- DNA fragments are mixed with plasmids and become incorporated into them and joined using DNA ligase which forms phosphodiester bonds
- plasmids now have recombinant DNA
How do scientists see which bacterial cells have taken up plasmids with recombinant DNA?
- all the bacterial cells are grown on a medium containing the antibiotic ampicillin
- bacterial cells that have taken up plasmids will have the gene for antibiotic resistance, and so will survive
- however the plasmids that have closed up without incorporating the new gene will also survive
- marker genes are then used
Why do not all bacterial cells take up recombinant plasmids?
- only a few will take them up when mixed together
- some plasmids will close up again without incorporating the DNA fragment
- DNA fragments join together to form their own plasmid
What types of marker genes are used to identify whether a bacterial cell has taken up the gene?
- antibiotic resistance
- fluorescent proteins
- produce an enzyme whose action can be identified
What are the advantages and disadvantages of in vivo gene cloning?
advantages:
- can introduce genes into different organisms using vectors
- no risk of contamination
- very accurate
- precise as it cuts out specific genes
- produces transformed bacteria
disadvantages:
- slower than in vitro - relies on generation times
What are the advantages and disadvantages of in vitro gene cloning?
advantages:
- rapid
- does not require living cells
disadvantages:
- inaccurate
- inaccuracies in all samples produces
- risk of contamination with other biological materials
What are DNA probes and what is their function?
a short single-stranded section of DNA that has a label attached, either radioactive isotopes or fluorescent markers
- this allows DNA fragments to be detected which can be used to diagnose genetic disorders
How do DNA probes work?
- the order of nucleotides on the mutated gene is determined by DNA sequencing
- a fragment of DNA with complementary bases to the mutated portion is produced, and the probe is formed by radioactively/fluorescently labelling the DNA fragment
- probe is added to single-stranded portions of DNA for the person being screened (DNA treated with alkali to make it single-stranded)
- if the mutated gene is present, the probe will bind to the complementary base sequence (DNA hybridisation) which can be seen under x-ray film/ using a UV light
What is DNA hybridisation?
separated strands of DNA mix with a probe which binds to the complementary base pairs and hydrogen bonds form
How can probes be used to locate specific alleles of a gene?
- determine the base sequence of nucleotides using DNA sequencing
- a fragment of DNA is produced that is complementary to the mutant allele
- multiple copies of the probe are formed using PCR
- probes are made by attaching markers
- DNA of the patient is separated by heating it to break hydrogen bonds
- separated strands are cooled in a mixture containing probes
- DNA is washed to get rid of any unattached probes
- if the DNA contains the mutant allele, the probes will bind which can be detected using UV light
What is genetic screening and why is it important?
screening individuals to look for genetic mutations using DNA probes
- can be used for people who may have a family history of a disease, allows them to see if they are a carrier and parents at risk of having affected children can obtain advice
- can be used to detect oncogenes which lead to cancer, mutations of both alleles must be present to inactivate tumour suppressor genes so people with one mutated gene are more at risk , they can they make informed decisions about their lifestyle and future treatments
What is personalised medicine?
- doctors can provide advice or care based on a patients specific genotype
- by genetically screening patients, the dose of a drug to produce the desired outcome can be used which saves money that would be wasted on overprescribing a drug
- avoids medication that could cause harm or avoid raising false hopes
What is genetic counselling?
- advice and information are given that enable people to make personal decisions about themselves and their offspring based on their genotypes
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