Chapter 21 - Recombinant DNA Technology Flashcards
Define recombinant DNA technology
A general term covering processes by which genes are manipulated, altered or transferred from organism to organisms
Define genetically modified organisms
An organisms that has had its DNA altered as a result of recombinant DNA technology
Give and briefly describe the five stages of recombinant DNA technology
- Isolation: Production of DNA fragments with the gene
- Insertion: Into a vector
- Transformation: Introduction into host cell
- Identification: Of host cells that have the DNA using gene markers
- Growth/cloning: Bt large scale cultures
What are the processess in which we can produce isolated DNA fragments?
- Conversion of mRNA to cDNA using reverse transcriptase
- Using restriction endonuclease to cut fragments with the gene
- Creating a gene with a gene machine
How can we use reverse transcriptase to produce genes for RDNAT?
- Cell that produces the protein is selected
- These cells have large quantities of mRNA needed
- Reverse transcriptase used to make cDNA single strand from RNA
- DNA polymerase builds up complementary nucleotides using cDNA as template to produce DNA with target gene
Define complementary DNA
DNA that is made from messenger RNA in a process that is the reverse of normal transcription
What does reverse transcriptase do?
Catalyse the production of DNA from RNA
How do restriction endonucleases produce genes for RDNAT?
Enzymes cut palindromic portions of DNA to leave sticky ends. The gap created is where the gene can be inserted
Define palindromic sequence
A section of DNA which reads the same whether read from 5’ to 3’ or 3’ to 5’ direction
How does the gene machine manufacture genes?
- Sequence of nucleotides found from protein->mRNA->DNA
- Sequence fed to computer
- Checks for safety/ethics
- Oligonucleotides assembled in sequence
- Gene replicated using PCR which forms DNA
Why is the gene machine beneficial to prokaryotes?
No introns are produced
How are genes cloned “in-vivo?”
- Isolated DNA fragments and plasmid cut with same restriction enzyme
- DNA inserted and complementary to sticky ends
- Fragments incubated with plasmids and DNA ligase which forms phosphodiester bonds
- RDNA molecule made
How are vectors stimulated to take up RDNAT plasmids?
Electroporation
How does electroporation work?
- Use of calcium ions and temperature change of 0-40 degrees celsius
- Increases permeability of membranes
Why don’t all cells take up RDNAT plasmids in electroporation?
- Only some take up the plasmids when mixed together
- Some plasmids will close up again without the DNA fragment
- DNA fragments form their pwn plasmid
What are marker genes?
A separate gene used to identify whether a gene has been taken up bay bacterial cells
Give three reasons as to why marker genes are identifiable
- Antibotic resistance
- Fluorescent
- Produces enzyme who’s action is identifiable
Summarise the PCR
- DNA sample, primers, DNA polymerase and nucleotides mixed
- Heated to 95 to break hydrogen bonds
- Cooled to 55 so primers can bind
- Heated to 70 for Taq polymerase
- Polymerase creates base pairing using nucleotides
- Repeated around 30 times for sufficient DNA
What are primers?
Short sequences of nucleotides that have set bases complementary to those of the two DNA fragments
What are the advantages of in vitro cloning?
- Rapid
- No living cells required
What are the advantages of in vivo cloning?
- Useful where we wish to introduce genes into organisms
- No contamination
- Accurate
- Cuts specific genes
- Produces bacteria that produce products
What is a DNA probe?
Short single stranded length of DNA that has a label, making it identifiable
Describe two types of DNA probes
- Radioactive nucleotides with a 32P isotope
- Fluorescent probes which emit light under certain conditions
How are DNA probe used to identify alleles?
- DNA probe created complementary to DNA of the allele
- DNA separated
- DNA mixed with probe during DNA hybridisation
- Site at the probe binding is identified by the label on the probe
What is DNA hybridisation?
When a section of DNA or RNA is combined with a single strand of DNA which had complementary bases
Why is DNA heated in DNA hybridisation?
To separate the two strands of DNA
What are VNTRs?
DNA base sequences that do not code and are different in length and number in all individuals
State the five stages of genetic fingerprinting
Extraction Digestion Separation Hybridisation Development
How does gel electrophoresis work?
- DNA fragments placed in agar gel
- Voltage applied
- Large moves slow, small moves quickly through the gel
- DNA probes analysed to determine position
Summarise the whole process of genetic fingerprinting
- DNA extracted
- DNA cut into blunt end fragments using restriction endonucelases
- DNA separated using gel electrophoresis and in alkali to create single strands. Southern blotting and fixed on to nylon with UV light
- DNA probes added which bind to VNTRs
- X-Ray film placed on nylon membrane to reveal patterns and location of DNA fragments
What is southern blotting?
In genetic fingerprinting where the pattern of DNA fragments are transferred to a nylon membrane
What are some uses of genetic fingerprinting?
- Crime solving
- Genetic relationships
- Medical Diagnosis
- Plant + Animal Breeding
