3.5 Genetic Modification and Biotechnology
Essential idea: Biologists have developed techniques for artificial manipulation of DNA, cells and organisms.
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3.5.U1 Gel electrophoresis is used to separate proteins or fragments of DNA according to size.
3.5.U1 Gel electrophoresis is used to separate proteins or fragments of DNA according to size.
Be able to:
Match restriction enzyme names to the bacteria in which they are naturally found.
Describe the role of restriction enzymes in nature and in biotechnology applications.
Contrast sticky vs. blunt ends.
Determine the number and size of DNA fragments after being exposed to restriction enzymes (both linear and plasmid DNA).
Demonstrate accurate use of a micro-pipette.
Explain the function and purpose of DNA electrophoresis.
Describe how and why DNA fragments separate during electrophoresis.
Outline the functions of the buffer, marker and loading dye in DNA electrophoresis.
In gel electrophoresis DNA fragments move in an electric field and separate from each other according to their sizes. The smallest fragments will move the farthest distance from the origin of the DNA sample.
separates charged molecules in an electric field according to size and charge
samples are in wells cast in a gel that’s immersed in a conducting fluid with applied electric field
molecules with negative and positive move in a opposite directions, so proteins are separated according to their charge
DNA molecules are too long to move therefore must be broken into smaller fragments
Stages of PCR
PCR occurs in a thermal cycler and uses variations in temperature to control the replication process via three steps:
Denaturation – DNA sample is heated to separate it into two single strands (~95ºC for 1 min)
Annealing – DNA primers attach to the 3’ ends of the target sequence (~55ºC for 1 min)
Elongation – A heat-tolerant DNA polymerase (Taq) binds to the primer and copies the strand (~72ºC for 2 min
3.5.U2 PCR can be used to amplify small amounts of DNA.
3.5.U2 PCR can be used to amplify small amounts of DNA.
Be able to:
State the function of the PCR.
Describe the selectivity of the PCR
Gel electrophoresis is a laboratory technique used to separate and isolate proteins or DNA fragments based on mass / size
Samples are placed in a block of gel and an electric current is applied which causes the samples to move through the gel
This causes samples of different sizes to separate as they travel at different speeds
PCR – polymerase chain reaction that makes large numbers of copies of DNA can use PCR for forensic investigations
PCR is used to copy specific DNA sequences by using a primer that binds
3.5.U3 DNA profiling involves comparison of DNA.
3.5.U3 DNA profiling involves comparison of DNA.
Be able to:
Outline the process of DNA profiling.
DNA profiling involves the use of PCR and gel electrophoresis of DNA
sample of DNA is obtained
copied DNA is split into fragments using restriction endonucleases
fragments separated by gel electrophoresis
compared to see which bands are the same and which are different
Forensics: DNA fingerprinting for criminal cases
collect samples of DNA-containing substance (blood, semen)
from suspect
from crime scene
amplify quantity using PCR if necessary
add endonucleases to produce DNA RFLPs
use gel electrophoresis to separate RFLPs into DNA fingerprint
compare samples from crime scene and suspect for match
DNA Fingerprinting Animation
DNA Fingerprinting Animation
DNA fingerprinting to determine identity
collect samples of DNA-containing substance (blood, semen)
from children
from suspected father
amplify quantity using PCR if necessary
add endonucleases to produce DNA RFLPs
use gel electrophoresis to separate RFLPs into DNA fingerprint
compare samples from children and suspected father for matching RFLP patterns
3.5.U4 Genetic modification is carried out by gene transfer between species.
3.5.U4 Genetic modification is carried out by gene transfer between species.
A gene determines a particular trait by encoding for a specific polypeptide in a given organism. Because the genetic code is (almost) universal, an organism can potentially express a new trait if the appropriate gene is introduced into its genome. The transfer of genes between species is called gene modification, and the new organism created is called a transgenic. When genes are transferred between species, the amino acid sequence translated from them is unchanged – the same polypeptide is produced. Genes are transferred from eukaryotes –> bacteria
Example – goats produce that secrete milk containing spider silk protein – spider silk is immensely strong but spiders could not be used to produce it commercially
GM crops
Example of GM crops – golden rice involved the transfer of three genes
3.5.U5 Clones are groups of genetically identical organisms, derived from a single original parent cell.
3.5.U5 Clones are groups of genetically identical organisms, derived from a single original parent cell.
Be able to:
Contrast sexual and asexual reproduction.
Define clone and cloning.
Describe different ways in which natural clones can arise.
Clones are groups of genetically identical organisms or a group of cells derived from a single original parent cell
3.5.U6 Many plant species and some animal species have natural methods of cloning.
3.5.U6 Many plant species and some animal species have natural methods of cloning.
Clones are groups of genetically identical organisms that are derived from a single original parent cell. Cloning is the process through which these identical organisms are produced. The size of clones can range from two (i.e. identical twins) to hundreds (i.e. crops of commercially grown plants).
Organisms that reproduce asexually, which results in genetically identical organisms. Sexual reproduction produces genetically different organisms as a result of gamete fusion.
Binary Fission
The parent organism divides equally in two, so as to produce two genetically identical daughter organisms
This method of cloning occurs in Planaria (flatworms) but is also common to bacteria and protists (e.g. euglena, amoeba)
Budding
Cells split off the parent organism, generating a smaller daughter organism which eventually separates from the parent
This method of cloning occurs in Hydra but is also common to many species of yeast
Fragmentation
New organisms grow from a separated fragment of the parent organism
This method of cloning is common to starfish and certain species of annelid worms
Parthenogenesis
Embryos are formed from unfertilised ova (via the production of a diploid egg cells by the female)
This method of cloning occurs in certain species of insect, fish, amphibians and reptiles
Plant Cloning Methods
Plants have the capacity for vegetative propagation, whereby small pieces can be induced to grow independently
This is because adult plants possess meristematic tissue capable of cellular differentiation (totipotent)
Virtually all types of roots and shoots are capable of vegetative propagation
Garlic and onion bulbs are modified plant leaves – all the bulbs in a group are genetically identical
Underground stems (e.g. potato tubers) can form new plants which are genetically identical to the parent plant
Certain plants can form horizontal stems called runners (or stolons) that grow roots and develop into clones
Some plants (mainly algae, mosses and ferns) can reproduce asexually by producing spores
Spores are also produced by certain types of bacteria and fungi
Human Cloning Methods
Even human beings are capable of creating genetic clones through natural means
Identical twins (monozygotic) are created when a fertilised egg (zygote) splits into two identical cells, each forming an embryo
Non-identical twins (dizygotic) are created when an unfertilised egg splits into two cells and each is fertilised by a different sperm
Identical twins will be clones of one another (genetically identical), while non-identical twins will share 50% of the same DNA
3.5.U7 Animals can be cloned at the embryo stage by breaking up the embryo into more than one group of cells.
3.5.U7 Animals can be cloned at the embryo stage by breaking up the embryo into more than one group of cells.
Be able to:
Describe the process of reproductive cloning via embryo splitting.
Outline example of cloning animal embryos via natural and artificial embryo splitting.
3.5.U8 Methods have been developed for cloning adult animals using differentiated cells.
3.5.U8 Methods have been developed for cloning adult animals using differentiated cells.
Be able to:
Describe the process of cloning via somatic cell nuclear transfer.
Cloning farm animals
differentiated mammary cells extracted from parent sheep; grown in nutrient-deficient solution to stop the cell cycl
undifferentiated egg cells extracted from egg donor; nucleus removed and discarded
mammary cell placed next to enucleated egg cell
electric shock causes two cell membranes to fuse, and mitosis to trigger
mitotic division continues, producing embryo
embryo implanted into surrogate mother
after 5-month gestation, Dolly the lamb born with identical genotype to parent donating nucleus from mammary cell
3.5.A1 Use of DNA profiling in paternity and forensic investigations.
3.5.A1 Use of DNA profiling in paternity and forensic investigations.
Be able to:
List example sources of DNA that can be used in DNA profiling
DNA profiling involves the use of gel electrophoresis of DNA.
3.5.A2 Gene transfer to bacteria using plasmids makes use of restriction endonucleases and DNA ligase.
3.5.A2 Gene transfer to bacteria using plasmids makes use of restriction endonucleases and DNA ligase.
E. coli is used in gene technology because some of its DNA is found on plasmids (smaller circles of DNA). These plasmids can be removed and cleaved by restriction enzymes at target sequences. DNA fragments from another organism can also be cleaved by the same restriction enzyme, and these pieces can be added to the open plasmid and spliced together by ligase. The recombinant plasmids formed can be inserted into new host cells and cloned. When genes are transferred between species, the amino acid sequence of polypeptides translated from them is unchanged because the genetic code is universal.The basic technique in gene transfer is outlined below.
3.5.A3 Assessment of the potential risks and benefits associated with genetic modification of crops.
3.5.A3 Assessment of the potential risks and benefits associated with genetic modification of crops.
Possible benefits
benefits include more specific breeding than with traditional methods
faster than traditional methods
selective breeding cannot produce desired phenotype
increase productivity of food production
less use of echemicals (e.g. pesticides)
food production possible in extreme conditions
less expensive drug preparation (e.g. pharmaceuticals in milk)
human insulin engineered some no allergic reactions
may cure genetic diseases
Possible harmful effects
some gene transfers are regarded as potentially harmful to organisms especially animals
release of genetically engineered organisms into environment
can spread and compete with natural occurring varieties
some engineered genes can also cross species barriers
technological solution when less than their methods may bring similar benefits
reduces genetic variation
3.5.A4 Production of cloned embryos produced by somatic-cell nuclear transfer.
3.5.A4 Production of cloned embryos produced by somatic-cell nuclear transfer.
Be able to:
Outline the production of Dolly the sheep using somatic cell nuclear transfer.
Somatic cell nuclear transfer is a method by which cloned embryos can be produced using differentiated adult cells. 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
Artificial Embryo Twinning
Low-tech version of cloning which mimics the natural processes of producing identical twins
An egg is fertilized by sperm
In the early stages of its formation, the embryo is split into individual cells
Then placed in a surrogate to grow to a full organism
The resulting offspring produced are identical
Somatic Cell nuclear Transfer
Egg cell nucleus is removed and replace by the nucleus of a somatic cell (e.g. skin cell)
Under certain conditions, the egg begins to replicate as though it was a fertilized embryo
3.5.S1 Design of an experiment to assess one factor affecting the rooting of stem-cuttings.
3.5.S1 Design of an experiment to assess one factor affecting the rooting of stem-cuttings.
A stem cutting is a separated portion of plant stem that can regrow into a new independent clone via vegetative propagation
All stems possess nodes, from which a leaf, branch or aerial root may grow – the region between nodes are called internodes
Stem cuttings are typically placed in soil with the lower nodes covered and the upper nodes exposed
Stem cutting is a common method employed to rapidly propagate plant species (including sugar cane, grapes and roses)
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
3.5.S2 Analysis of examples of DNA profiles.
3.5.S2 Analysis of examples of DNA profiles.
Be able to:
Analyze a DNA profile to determine relatedness or forensic guilt.
3.5.S3 Analysis of data on risks to monarch butterflies of Bt crops.
3.5.S3 Analysis of data on risks to monarch butterflies of Bt crops.
Bacillus thuringienis (Bt) cor, is a transgenic plant, contains Bt gene encording CryIAb protein toxin
Developed to suppress the European corn borer larvae (i.e. target organism) that feeds on the pollen grains that accumulate on corn leaves
Also has unintended effects on species (e.g. monarch butterfly larvae; non-target organism) beyond transgenic field borders
Feeds on milkweed plant but also ingests wind-dispersed pollen that has landed on the leaves
The pollen contains the expensed insecticidal toxins of th eBt gene that kills monarch larva.
Transgenic Bt corn pollen has a negative effect on effect on monarch butterfly larvae feedling next to Bt corn fields
Both concentrations of pollen (i.e. amount of Bt gene toxin) decreased larval survival rates
1,300 grains/cm2 had lower survival rate than the 135 grains/cm2
Therefore, higher concentrations of Bt gene toxin decreased larval survival rates
Wind-dispersed pollen containing Bt gene toxin can cause larval mortality
e.g. Event 176 pollen causes larval mortality in non-Bt plants
Biotechnology in the world today- the CRISPR
Biotechnology in the world today- the CRISPR
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