Ch 5: Biotechnology and Genetic Techniques Flashcards
What is biotechnology?
the use of living things to make new products or systems
What are restriction endonucleases?
the enzyme restriction endonucleases that cuts the DNA at a specific restriction site, the pieces cut are known as restriction fragments
- these enzymes occur naturally in bacteria where they cleave foreign DNA that enters from invading viruses like an immune system, they are named according to the bacteria from which they are derived
How is DNA cut?
the enzyme, restriction endonuclease recognises a specific sequence of 4-8 nucleotides base pairs, restriction enzymes bind to their restriction site and cut the double stranded DNA at that point.
What are the different types of ends produced from cutting DNA?
sticky ends:
- the end of a DNA fragment that is created following a cleavage by a restriction enzyme that cuts DNA at different positions on each strand. DNA is then able to be joined to other exposed end fragments of DNA with complementary sticky ends to their recognition sites
blunt ends:
- end of a DNA fragment that is created following a cleavage by a restriction enzyme that cuts DNA at the same point on both strands. DNA is able to be joined to any other blunt end fragment but tends to be nonspecific because there is no sticky ends as recognition sites
How is recombinant DNA produced?
- Gene of interest is cut out with restriction enzyme from foreign DNA, plasmid cut open with same enzyme
- Gene inserted into plasmid, usually stick ends, using complementary base pairing
- Two fragments of DNA are sealed by DNA ligase by forming a phophodiester bond between the 3’ hydroxyl end of one nucleotide with the 5’ phosphate end of another nucleotide
- Recombinant DNA plasmids are cloned/many copies produced
- Recombinant plasmids are inserted into new host cells/viruses/bacteria/yeast. A vector can be used to transport the wanted/foreign DNA into a host organism. Inserted by shooting/spraying/microencapsulation/ heat treatment
What is ligation?
reassembling of DNA fragments produced using restriction enzymes
What is polymerase chain reaction?
PCR is a cyclical reaction in which DNA polymerase is used to copy a DNA template, making millions of copies of the same piece of DNA
What are the steps of PCR?
- 4 nucleotide bases, template sample of DNA, heat resistant DNA polymerase Taq and single stranded DNA primers are placed in a DNA thermocycler
- 95C: denaturation of DNA, separating strands by breaking hydrogen bonds
- 50-60C: annealing, primers bind onto their complementary sequence
- 72C: DNA polymerase enzyme begins to move along the template DNA, starting from the primer and adding nucleotides according to the complementary base pairing rules
- thermocycler heats to 95C and the next round of strands separate, binding of primers and DNA replication begins
- 20-40 cycles are used to amplify a sample (1 million copies)
What is the process of gel electrophoresis?
- the agarose gel is melted and poured into a flat mould to cool, wells are created by placing a plastic comb into the gel as it set
- gel is placed in a tray filled with buffer solution and positive and negative electrodes are attached at each end, DNA has an overall negative charge due to its phosphate group backbone
- the gel acts like a sponge, the DNA strands move through when the electric current runs, repelled by the negative electrode and moves towards the positive electrode. Smaller strands can move through the gel faster which separates the strands by size
- ethidium bromide or another fluorescent DNA-binding dye is added to the agarose gel to view the separated DNA fragments. The dye binds to DNA and fluoresces under UV light, showing a pattern of bands that can then be photographed
- the position of the bands on the gel depends on the size and charge of the DNA fragments in each band, molecular size markers are used to determine the size of DNA
What is DNA profiling?
a process that is able to identify natural variations that exist within an individual’s genome, by using PCR and gel electrophoresis
What is microarray or gene probing?
used to investigate the level of genetic expression in cells by measuring the amount of mRNA present
What is the process of microarray?
- microarray slide is embedded with DNA of interest i.e. manufactured single-stranded DNA segments or DNA probes
- collect patient samples to compare differences in gene expression, samples are taken from a single subject and mRNA from these 2 cell types are isolated (denaturation, isolation, transcription)
- prepare cDNA samples: role of mRNA is to copy an active switched on gene however it is unstable and unreliable. So reverse transcriptase enzymes are used to convert mRNA into complementary DNA (cDNA). cDNA is more stable and less likely to degrade. The two types of cDNA collected are labelled with fluorescent dyes to distinguish them from each other
- add cDNA to microarray: the cDNA mixture is added to the microarray slide. WHen the cDNA sample is added to the microarray slide it will hybridise (bind) with single stranded DNA probes embedded in the slide. The slide is washed to remove cDNA that didn’t bind with DNA probes. The colours are scanned and analysed.
What is DNA sequencing and what is the process?
identifies the exact nucleotide sequence of DNA fragments
- denaturation of DNA strand at 95C
- annealing, 60C and primers attach to complementary DNA sequence
- extension: 72C Taq polymerase attaches to primer and begins attaching free nucleotides. Extension stops when a dideoxynucleotide (didNTP) randomly joins a fragment as it is missing a hydroxyl group. All possible terminations are produced
- gel electrophoresis: fragments are separated by size, didNTP fluoresce under UV light
- electropherogram is produced, height = intensity of the fluoresce (how often same termination appears)
- nucleotide sequence is determined by the colour code
What is the process of gene cloning using plasmids?
it is an alternative to PCR, utilising bacterial plasmids to produce many copies of a gene
- plasmids are extracted from bacteria by rupturing the cell wall
- the same restriction enzyme is used to cut the plasmid DNA and the DNA of the gene to be inserted so that both pieces have complementary sticky ends
- DNA ligase binds the foreign DNA fragment into the plasmid DNA. After binding, the DNA fragment becomes a permanent part of the recombinant plasmid
- the recombinant plasmids are added to a bacterial culture. They are taken up by some bactera, in which they replicate as in the normal process of growth and division, bacteria replicate the plasmid and thus numerous copies of the incorporated foreign DNA are made
transformation: process of bacteria taking up DNA, some seal up without taking up the plasmid
What is antibiotic selection?
plasmid DNA often contains genes for resistance to an antibiotic i.e. ampicillin. Bacteria that have been transformed with the plasmid are able to grow and multiply on a medium that is supplmented with ampicillin because they are resistant to it. This bacteria are selected and grown in a culture, the plasmids isolated and analysed
What is a vector?
an organism that transports genes from one host to another
Compare plasmid, viral and liposome vectors
plasmid vectors: purifying recombinant plasmids can be inserted into a new organism directly however plasmid DNA is not very stable and hence the process is not efficient
viral vectors: desired genes are inserted into viral DNA/RNA and the virus then inserts this gene into the target cells, genes that cause disease symptoms must be removed yet they can still disrupt gene regulation and cause cancer and the human immune system attacks viruses reducing their survival
liposome vectors: liposomes are small spheres surrounded by a phopholipid bilayer membrane, this membrane can fuse with other membranes delivering the contents of the liposome into the cell. Liposomes can be made artificially and are designed to have molecules attached to their surface that can be recognised by specific types of cells in the host organism, thus when it is inserted into a foreign organism, it be targeted to reach specific cells and the gene of interest be inserted
How are individuals differentiated?
short tandem repeats (STRs - sequences 2-5 bases) and variable nucleotide tandem repeats (VNTR - more than 5 bases sequence) are sections of non-coding DNA that are repeated several times. The repeat is present in all members of a population but the number of repeats varies between individuals. Each individual usually has 2 alleles for each STR, one from each homologous chromosome. DNA profiling identifies people based on differences in length of their DNA repeats for a large number of individual STRs. As every individual has their own unique no. of repeats, this forms the basis of identification
What are transgenic organisms?
incorporation of cloned DNA into the genomes of multicellular organisms, usually from another species
