Genetic engineering and Biotechnology Topic 4.4 “Or how I stopped worrying and learned to love the sheep.”

polymerase chain reaction (PCR) scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA.

PCR POLYMERASE CHAIN REACTION Used in research Study a particular sequence Need of identical copies method of cloning—lots of time and work

It uses enzymes to replicate DNA Enzyme is isolated from bacteria and yeast. KARY MULLIS-developed in 1983 Nobel prize

Principle of PCR Desired DNA is heated---break the Hydrogen bond Two strand separate Primes are added to start Replication Mixture is cooled. Primers bind to the original to ssDNA. Nucleotides and thermostable DNA- Polymerase is added Nucleotides will bond with exposed bases of the ssDNA The original strand has formed a new CDNA. Cycle is repeated

Gel Electrophoresis Gel electrophoresis is a technique used to separate fragments of DNA.Gel electrophoresis is a technique used to separate fragments of DNA. Separates fragments as a function of size and charge.Separates fragments as a function of size and charge. Most types use Agarose to separate fragments.Most types use Agarose to separate fragments. Agarose is a porous gel. It can allow the passage of molecules through, however, larger molecules move more slowly through it since they cannot squeeze through the pores as easily as smaller molecules.Agarose is a porous gel. It can allow the passage of molecules through, however, larger molecules move more slowly through it since they cannot squeeze through the pores as easily as smaller molecules. Electrophoresis Apparatus

Electrophoresis Technique An agarose gel is casted with several holes called wells at one end.An agarose gel is casted with several holes called wells at one end. The gel is placed in an electrophoresis box which is filled with an electrolyte buffer solution.The gel is placed in an electrophoresis box which is filled with an electrolyte buffer solution. Samples of digested DNA are placed in the wellsSamples of digested DNA are placed in the wells Electrical leads are attached to the ends of the box creating an electrical potential across the apparatus.Electrical leads are attached to the ends of the box creating an electrical potential across the apparatus. Because DNA has a negative electrical charge. It is "pulled" towards the positive side of the apparatus.Because DNA has a negative electrical charge. It is "pulled" towards the positive side of the apparatus. Also, since the smaller molecules travel faster through the agarose. Over time this separates the various sized fragments of DNA.Also, since the smaller molecules travel faster through the agarose. Over time this separates the various sized fragments of DNA. The gel is then removed and stained for DNA. This results in a gel which shows several bands of stained DNA.The gel is then removed and stained for DNA. This results in a gel which shows several bands of stained DNA.

Finished Gel

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DNA Fingerprinting DNA profiling is also known as DNA fingerprinting DNA is now a powerful tool in identification. Based on the fact that the amount of "junk DNA" differs uniquely between individuals. Structural genes are often separated by large regions of repeating base pairs. The number of these repeats is unique to an individual. Therefore when DNA from a person is cut with a restriction enzyme, the length of the fragments will be unique to an individual.

DNA Fingerprinting Contd… This will therefore produce a unique banding pattern following a gel electrophoresis.This will therefore produce a unique banding pattern following a gel electrophoresis. This test is highly accurate, and the probability of another individual possessing an identical banding pattern is estimated as around 1:14,000,000,000.This test is highly accurate, and the probability of another individual possessing an identical banding pattern is estimated as around 1:14,000,000,000.

Crime scene Small amounts of DNA Amplify the DNA-PCR Two strands are separated Restriction enzymes- endonucleases used to cut Sections will differ in size and charge Separated by Gel electrophoresis Pattern of stripes and bands determined by the sequence of the bases

DNA Fingerprinting

Application a. Paternity profiling -each band shown on the DNA Profile of a child must correspond with a band of the father or the mother. b. Forensic investigation- -Compare DNA from the suspect with DNA from the crime scene sample (blood, hairs, semen c. Using relative’s DNA to determine the identity of a victim -to determine the identity of the remains of dead people.

For Eg., Tsar of Russia and his family was shot during the Russian revolution and bodies were shown to prove it. By taking blood samples of distant relatives of the Romanovs DNA patterns could be established Conclusion- Bodies were likely to be the Romanov family

see fingerprinting for some ideas of problems when using DNA profiling as evidence.

Human genome project commitment undertaken by the scientific community across the world. International Human Genome Organisation –is an excellent example of how collaboration of scientists across the world can benefit all of us. To determine the locations and structure of all genes in the human chromosomes Data was pooled

Suggested in started (3x10 9 base pairs) in human DNA In 2003 the sequencing of the human DNA was 99.9% complete. Mapping of genes-listing and finding the locus of each human gene

Outcomes of having sequenced the entire human Genome An improved understanding of many genetic disease The production of medicines (based on DNA sequences) to cure and/ or genetic engg. To remove the gene which causes the diseases To determine fully which genetic diseases any individual is prone to ( genetic screening leading to preventive medicine)

Research into a particular disease can focus on only gene(s) Provide more info about evolutionary paths by comparing similarities and differences in genes between species. Info is valuable BUT it could be abused.— insurance companies, prospective employer and society faces the challenge of coming to terms with the ethical issues

Genetic engineering Deliberate manipulatipn of genetic material. This is possible due to- Universal genetic code Transfer the genetic material from one species to another Introduce human gene for making insulin into a bacterium The bacterium produces human protein insulin.

Gene transfer Requires following elements A vector-Plasmid-to carry the gene into the host A host cell Restriction enzymes DNA ligase

Restriction Enzymes Restriction enzymes are compounds first isolated in the 1970'sRestriction enzymes are compounds first isolated in the 1970's They function by selectively cutting DNA at specific sequencesThey function by selectively cutting DNA at specific sequences

Restriction Enzymes These cuts usually occur in the following forms.These cuts usually occur in the following forms. The cut can be made straight across a base- pair sequence resulting in a "Blunt End“The cut can be made straight across a base- pair sequence resulting in a "Blunt End“ The cut can be made in an offset manner leaving exposed nucleotide sequences. These exposed sequences are called "Sticky Ends"The cut can be made in an offset manner leaving exposed nucleotide sequences. These exposed sequences are called "Sticky Ends" Blunt End Sticky end

Gene Splicing The presence of sticky ends allows segments of DNA to be joined together.The presence of sticky ends allows segments of DNA to be joined together. Since DNA strands which have been cut by the same restriction enzyme can easily bond together according to base pairing rules. Since DNA strands which have been cut by the same restriction enzyme can easily bond together according to base pairing rules.

Recombinant DNA

Gene Splicing contd.. This allows for genes to be "cut & pasted" between organisms. This can be seen with production of human insulin.This allows for genes to be "cut & pasted" between organisms. This can be seen with production of human insulin. The DNA sequence of insulin is identified and cut out using a restriction enzyme.The DNA sequence of insulin is identified and cut out using a restriction enzyme. A plasmid from E. coli is removed and cut open using the same restriction enzymeA plasmid from E. coli is removed and cut open using the same restriction enzyme Since both fragments have complimentary sticky ends and the gene for human insulin is integrated into the plasmidSince both fragments have complimentary sticky ends and the gene for human insulin is integrated into the plasmid The plasmid is then reinserted into a bacterial cell. This cell will produce insulin and is cultured. Human insulin can now be extracted and provided to diabetics.The plasmid is then reinserted into a bacterial cell. This cell will produce insulin and is cultured. Human insulin can now be extracted and provided to diabetics.

Two examples-GM crops and animals GMO-Genetically manipulated organisms Called transgenic organisms

Flavr Savr 1994-first GM food was sold commercially. Tomato was altered---stay fresh longer Gene was introduced to block the enzyme which cause rotting No longer available

Bt corn G M Maize. A gene from Bacillus thuringiensis (Bt) Incorporated into maize Plants produce a toxin that makes them resistant to insects Bt crops are grown in US

Bt Corn European corn borer, ECB found also in US The ECB through stems and leaves of the corn plant and will damage vascular bundles disrupt the transport of water and nutrients through the plant. It can also weaken the stems and leaves so that the plant or leaves may break Bt corn is already in commercial use.

benefits of Bt corn The damage caused by the ECB is much reduced. Bt corn is slightly more expensive, but the difference is less than one extra application of insecticide. Non-Bt corn needs to be checked often for signs of ECB - less checking needed for Bt corn.

Less insecticide needed means less impact on the environment and lower health risks for the worker(s) Seems to reduce the infection with fungus so mycotoxin (poisons produced by fungi) levels are lowered. Mycotoxins are difficult to remove by cooking/freezing and may go into the food chain and be found in meat of animals which ate the infected corn. Mycotoxins can be a hazard to human and animal health.

harmful effects of bt corn Will also kill some other insects (though many are not affected). Insects may develop resistance to Bt toxin because they are exposed to it all the time Resistant insects also make Bt spray useless as insecticide (Bt spray is considered to be relatively safe for humans and the environment).

It is difficult to prevent pollen (with the Bt gene) from travelling outside the field where the Bt corn is grown -it may fertilise non-Bt corn e.g. organically grown corn which can then no longer be sold as organic corn. -it may fertilise wild relatives and make them more resistant to insects and have them dominate the niche they live in. -This would result in loss of biodiversity.

examples GM mice--- to study the disease polio Possible treatment and prevention. Golden Rice Rice is major part of their diet Suffer from Vit A deficiency---lead to blindness Rice store Kit A in leaves but not in rice grains By adding genes from daffodills and from bacterium Plant stores a precursor of Vit A in the grains--- yellow color

New kind of rice is now produced One gene from maize and bacterium 20 times more of beta carotene compared of Golden Rice. valuable source of Vit A opposed by environmentalist, and anti- globalisationists.

Cloning

clone Group of genetically identical organisms or group of cells derived from a single cell Using differentiated cells is mostly somatic cells nuclear transfer-SCNT. Reproductive cloning Therapeutic cloning

Cloning: What it is Cloning is the process of making a genetically identical organism through nonsexual means. It has been used for many years to produce plants (even growing a plant from a cutting is a type of cloning). It has been used for many years to produce plants (even growing a plant from a cutting is a type of cloning). Animal cloning has been the subject of scientific experiments for years, but garnered little attention until the birth of the first cloned mammal in 1997, a sheep named Dolly. Animal cloning has been the subject of scientific experiments for years, but garnered little attention until the birth of the first cloned mammal in 1997, a sheep named Dolly. Since Dolly, several scientists have cloned other animals, including cows and mice.Since Dolly, several scientists have cloned other animals, including cows and mice. The recent success in cloning animals has sparked fierce debates among scientists, politicians and the general public about the use and morality of cloning plants, animals and possibly humansThe recent success in cloning animals has sparked fierce debates among scientists, politicians and the general public about the use and morality of cloning plants, animals and possibly humans Dolly, the first mammal clone

Dolly: A Mammal Clone DollyDolly In 1997, cloning was revolutionized when Ian Wilmut and his colleagues at the Roslin Institute in Edinburgh, Scotland, successfully cloned a sheep named Dolly. Dolly was the first cloned mammal. In 1997, cloning was revolutionized when Ian Wilmut and his colleagues at the Roslin Institute in Edinburgh, Scotland, successfully cloned a sheep named Dolly. Dolly was the first cloned mammal. Wilmut and his colleagues transplanted a nucleus from a mammary gland cell of a Finn Dorsett sheep into the enucleated egg of a Scottish blackface ewe.Wilmut and his colleagues transplanted a nucleus from a mammary gland cell of a Finn Dorsett sheep into the enucleated egg of a Scottish blackface ewe. The nucleus-egg combination was stimulated with electricity to fuse the two and to stimulate cell division.The nucleus-egg combination was stimulated with electricity to fuse the two and to stimulate cell division. The new cell divided and was placed in the uterus of a blackface ewe to develop. Dolly was born months later.The new cell divided and was placed in the uterus of a blackface ewe to develop. Dolly was born months later.

Clone a MIMI mouse cloning/

Reproductive cloning Creates a new cloning Dolly the sheep Dolly is known as SCNT it is theoretically possible to apply the same technique to cloning other species. Horses are an example of a species cloned successfully, but attempts with several other species have been less successfull.eg Eg., mare and her cloned foal

Therapeutic cloning Involves stem cell research Human embryos are produced and allowed to grow for few days into small of ball of cells These cells are not specialised but when SCNT is used the cells can grow into any different specialised tissues. Other sources of stem cells from umbilical cord or cells from aborted fetuses.

Aims for cell therapy Used –Parkinson’s disease Bone marrow transplants, skin cells for burn victims Grow new corneas

Ethical issues of Therapeutic cloning in humans Arguments in favor of therapeutic cloning focus on: the ability to cure serious diseases with cell therapy: currently leukemia and in the future possibly cancer and diabetes.

Some of the concerns raised about therapeutic cloning relate fears of it leading to reproductive cloning use of embryonic stem cells involves the creation and destruction of human embryos (although it is possible to use embryos left over from IVF treatment which would be destroyed anyway) embryonic stem cells are capable of many divisions and may turn into tumors. Ethical aspects of cloning are difficult to discuss since a lot of the benefits are currently not yet realised. They are potential benef its.