3.5 Flashcards
Stages of PCR
Denaturation
Annealing
Elongation
What are clones?
are groups of genetically identical organisms, derived from a single original parent cell
What is PCR denaturation
DNA sample is heated to separate it into two single strands (~95ºC for 1 min)
What is PCR annealing
DNA primers attach to the 3’ ends of the target sequence (~55ºC for 1 min)
What is PCR elongation
A heat-tolerant DNA polymerase (Taq) binds to the primer and copies the strand (~72ºC for 2 min)
What is PCR?
polymerase chain reaction (PCR) - an artificial method of replicating DNA under laboratory conditions
Once large quantities of DNA have been created, other laboratory techniques are used to isolate & manipulate the sequences
What is PCR used for?
- to amplify large quantities of a specific sequence of DNA from an initial minute sample
- Each reaction cycle doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies (2^30)
What is Gel electrophoresis?
a laboratory technique used to separate and isolate proteins or DNA fragments based on mass / size
How does gel electrophoresis work?
Samples are placed in a block of gel and an electric current is applied which causes the samples to move through the gel
Smaller samples are less impeded by the gel matrix and hence will move faster through the gel
This causes samples of different sizes to separate as they travel at different speeds
DNA seperation procedure by gel electrophoresis
DNA may be cut into fragments using restriction endonuclease – different DNA samples will generate different fragment lengths
Fragments separate bc DNA is negatively charged due to the presence of a phosphate group (PO43–) on each nucleotide
DNA samples are placed into an agarose gel and fragment size calculated by comparing against known industry standards
Specific sequences can be identified by incorporating a complementary radiolabelled hybridisation probe, transferring the separated sequences to a membrane and then visualising via autoradiography (Southern blotting)
Steps of gene transfer
- Isolation of gene and vector (by PCR)
- Digestion of gene and vector (by restriction endonuclease)
- Ligation of gene and vector (by DNA ligase)
- Selection and expression of transgenic construct
what is a vector?
a DNA molecule that is used as a vehicle to carry the gene of interest into a foreign cell
What are commonly used as vectors? and why?
Bacterial plasmids
bc they are capable of autonomous self-replication and expression
What are other types of vectors
modified viruses and artificial chromosomes
Step 1 of gene transfer
Step 1: Isolating gene and vector
DNA isolated from cells by centrifugation – whereby heavier components such as nuclei are separated
gene of interest can then be specifically amplified via (PCR)
Gene sequences can also be generated from mRNA using reverse transcriptase – these DNA sequences (cDNA) lack introns
Step 2 of gene transfer
Step 2: Digestion with Restriction Enzymes
gene of interest & vector must be cut w restriction enzymes at specific recognition sites
Restriction enzymes cleave the sugar-phosphate backbone to generate blunt ends or sticky ends (complementary overhangs)
Scientists will often cleave the vector & gene w 2 different ‘sticky end’ restriction endonucleases (double digestion) to ensure the gene is inserted in the correct orientation & to prevent the vector from re-annealing without the desired insert
Step 3 of gene transfer
Step 3: Ligation of Vector and Insert
gene of interest is inserted into a plasmid vector that has been cut w the same restriction endonucleases
This occurs bc the sticky ends of the gene & vector overlap via complementary base pairing
The gene & vector are then spliced together by the enzyme DNA ligase to form a recombinant construct
DNA ligase joins the vector & gene by fusing their sugar-phosphate backbones together with a covalent phosphodiester bond
GM Crops and Human Health
GM crops can be used to improve human nutritional standards, by incorporating genes for certain proteins, vitamin or vaccines
GM crops can be manufactured that lack common natural allergens or toxins
Inclusion/removal of certain genes could trigger unexpected adverse health reactions in some individuals
Currently, not all foods with GM components are labelled, making informed decisions of use difficult for consumers
GM Crops and Economic Consequences
GM crops can include genes to enable them to grow in a wider range of environments (e.g. drought / frost / salinity resistance)
GM crops can be manufactured to produce greater yields (crops can potentially grow larger and faster)
GM crops can include genes which slow the rate of spoiling, leading to longer shelf lives for GM foods
GM crops may possess resistance to certain viruses or produce toxins to pests (reducing need for the use of pesticides)
Herbicide resistant crops can be used to allow for the easier killing of weeds (which compete with crops for soil nutrients)
Overall, an improved yield, reduction in farming costs and ability to farm more land will provide an economic benefit to farmers
However, patent protection allows biotech companies to restrict the use of seeds and force farmers to pay high prices for use
GM Crops and Environmental Issues
The ability to farm a wider range of environments with GM crops will potentially reduce the need for associated deforestation
Also, the generation of pest-resistant crops means that less chemical insecticides will be released into the environment
However, GM crops could potentially reduce biodiversity in a region by competing with indigenous plant life
Furthermore, proteins or toxins produced by GM crops could negatively affect certain organisms within the ecosystem
Cross-pollination by GM crops could also result in the formation of herbicide-resistant weeds and grasses
GM crops with pest toxins could also accelerate the evolution of resistant pest species
What is Bt corn
a genetically modified maize that incorporates an insecticide producing gene from the bacterium Bacillus thuringiensis
Bt corn on butterflies
Concerns have been raised that the spread of Bt corn may also be impacting the survival rates of monarch butterflies
While monarch butterfly larva feed exclusively on milkweed, wind-borne pollen from Bt corn may dust nearby milkweeds
insecticide producing gene is lethal to certain types of larvae, particularly the European corn borer which would otherwise eat the crop
Effect of Bt Pollen on Survival Rates of Monarch Butterflies (Laboratory Conditions)
Some scientists suggested that these results may lack validity as they do not accurately reflect natural conditions:
There were higher amounts of Bt pollen on the leaves than would be found naturally (e.g. rain would diminish build up)
Larva were restricted in their diet (in the field, larva could feasibly avoid eating pollen dusted leaves)
A second study was conducted comparing the survivor rates of monarch butterflies based on proximity to Bt corn fields:
There was no significant increase in mortality when monarch larva were placed in or near an actual Bt corn field
From this it was concluded that exposure to Bt pollen poses no significant risk to monarch butterfly populations
What is Somatic cell nuclear transfer?
a method by which cloned embryos can be produced using differentiated adult cells
What is the process of SCNT?
Somatic cells are removed from the adult donor and cultured (these cells are diploid and contain the entire genome)
An unfertilised egg is removed from a female adult and its haploid nucleus is removed to produce an enucleated egg cell
The enucleated egg cell is fused with the nucleus from the adult donor to make a diploid egg cell (with the donor’s DNA)
An electric current is then delivered to stimulate the egg to divide and develop into an embryo
The embryo is then implanted into the uterus of a surrogate and will develop into a genetic clone of the adult donor
What are natural cloning mechanisms?
Binary fission
Budding
Fragmentation
Parthenogenesis
Plant cloning methods
vegetation propagation
What is a stem cutting?
a separated portion of plant stem that can regrow into a new independent clone via vegetative propagation
There are a variety of factors that will influence successful rooting of a stem cutting, including:
Cutting position (whether cutting occurs above or below a node, as well as the relative proximity of the cut to the node)
Length of cutting (including how many nodes remain on the cutting)
Growth medium (whether left in soil, water, potting mix, compost or open air)
The use and concentration of growth hormones (e.g. IAA, IBA and NAA promote the formation of adventitious roots)
Temperature conditions (most cuttings grow optimally at temperatures common to spring and summer)
Availability of water (either in the form of ground water or humidity)
Other environmental conditions (including pH of the soil and light exposure)
