Genetic engineering and biotechnology

Genetic engineering and biotechnology Topic 4.4

4.4.1Outline the use of PCR to copy and amplify minute quantities of DNA Sometimes DNA samples can be hard to obtain: • Using the technique called polymerase chain reaction (PCR), researchers are able to create vast quantities of DNA identical to trace samples. This process is also known as DNA amplification. • Many procedures in DNA technology require substantial amounts of DNA to work with, for example; • DNA sequencing • DNA profiling/fingerprinting • Gene cloning • Transformation • Making artificial genes A crime scene (body tissue samples) A single viral particle (from an infection) Fragments of DNA from a long extinctanimal

Separate Strands Separate the target DNA strands by heating at 98°C for 5 minutes Incubate Cool to 60°C and incubate for a few minutes. During this time, primers attach to single-stranded DNA. DNA polymerase synthesises complementary strands. Add Reaction Mix Add primers (short RNA strands that provide a starting sequence for DNA replication), nucleotides (A, T, G and C) and DNA polymerase enzyme. Repeat for about 25 cycles Repeat cycle of heating and cooling until enough copies of the target DNA have been produced. Steps in the PCR Process • The laboratory process called the polymerase chain reaction or PCR involves the following steps 1-3 each cycle:

Polymerase Chain Reaction Original DNA Sample • Although only three cycles of replication are shown here, following cycles replicate DNA at an exponentialrate and can make literally billions of copies in only a few hours. • The process of PCR is detailed in the following slide sequence of steps 1-5. Cycle 1 Cycle 2 Cycle 3

A DNA sample called the target DNA is obtained DNA is denatured (DNA strands are separated) by heating the sample for 5 minutes at 98C Primers (short strands of mRNA) are annealed (bonded) to the DNA Primer annealed

McGraw-Hill PCR animation Media showcase animation PCR song PCR song 2 The sample is cooled to 60°C. A thermally stable DNA polymerase enzyme binds to the primers on each side of the exposed DNA strand. This enzyme synthesises a complementary strand of DNA using free nucleotides. After one cycle, there are now two copies of the original sample. Nucleotides Nucleotides

Testing for GMOs using PCR • PCR primers do not bind unless there is a complementary sequence of nucleotides • A = T • T = A • G ≡ C • G ≡ C • One test for GM ingredients in food involves a primer that will only bind to the GM DNA. • If GM DNA is present, the PCR process will amplify the DNA • If no GM DNA is present, the PCR has no effect

Sample Wells Cathode Buffer Plastic frame Gel Anode Buffer solution 4.4.2In gel electrophoresis, fragments of DNA move in an electric field and are separated according to size • Gelelectrophoresis can be used to separate large molecules (including nucleic acids or proteins) on the basis of their size, electric charge, and other physical properties. • DNA is split into fragments using restriction enzymes • The DNA samples are placed in wells and covered with a buffer solution that gradually dissolves them into solution.

Wells: Holes created in the gel with a comb. Gel electrophoresis demo lab negative terminal Large fragments DNA solutions: Mixtures of different sizes of DNA fragments are loaded into each well. DNA markers: A mixture of DNA molecules with known molecular weights. They are used to estimate the sizes of the DNA fragments in the sample lanes. DNA fragments: The gel matrix acts as a sieve for the DNA molecules. Small fragments positive terminal Tray: Contains the set gel. • DNA has a negative charge because the phosphate groups are negatively charged. • The DNA fragments in the gel move through the gel towards the positive terminal of the electric field. • Smaller molecules move at a faster rate through the gel; longer fragments take longer to work through the small spaces in the gel. • Groups of DNA fragments can be seen as bands on the gel – usually seen with the help of a dye.

4.4.3 Gel electrophoresis of DNA is used in DNA profiling • DNA profiling (DNA fingerprinting) is a technique for genetic analysis, which identifies the variations found in the DNA of every individual. • The profile refers to the distinctive pattern of fragments which is used to identify an individual. • DNA profiling does not determine a base sequence for a sample but merely sorts variations in base sequences. • Only one in a billion (i.e. a thousand million) persons is likely to have an identical DNA profile, making it a useful tool for forensic investigations and paternity analysis

DNA profiling can be used for investigating: • the presence of a particular gene,(such as cystic fibrosis) in a family. • genetic relatedness of different organisms • e.g. checking on pedigree in stock breeding programs. • e.g. checking that captive populations of endangeredspecies are not inbred.

DNA profiling can be used for forensic purposes: • DNA fingerprints from tissue samples can be used as evidence in the same way traditional fingerprinting is used. • Which DNA fingerprint from the three suspects matches that of the tissue sample submitted as evidence? • Why would the DNA from the victim be included in this test? • The DNA from the victim must be excluded from the evidence. • In a paternity test, the DNA from the mother must also be included to exclude her contribution to the banding patterns in the child’s profile.

We expect 100% match as the cells left behind at the scene are the perpetrator’s cells

We expect 100% match as the cells left behind at the scene are the perpetrator’s cells The overlapping bands between victim and suspect indicate a close genetic relationship

No. Without a stronger match, the evidence is insufficient to convict the suspect. He should be released and a new suspect found. DNA evidence is being reviewed in many wrongful convictions.

In this case, determine which man (1 or 2) is the biological father of the child. Because the child inherits half its genetic material from each parent, any band that the child has not inherited from his mother, he must have inherited from his father.

1. Refer to the image on the right Which male (1 or 2) is the father of the child? Explain. 2. Refer to the image on the left Which suspect(1,2 or 3) was present at the crime scene? Explain.

“DNA is better at proving innocence than guilt.” • Discuss this statement

Textbook exercises: • Tiger book p164 • Look at figure 7 and determine the culprit • Tiger book p 166 • Answer question 1 on logarithmic scales in Biology • Tiger book p167 • Question 6

4.4.3 Outline three outcomes of the sequencing of the complete human genome • Completed in April 2003. • An international, collaborative effort to record • the entire base sequence of the human genome. • Also achieved the following: • Discovered the number and loci of all the genes (30k) in our genome – further research in diagnostics, treatment and pharmacology. • New proteins and their functions were discovered. • Comparisons between genomes of different species – evolutionary history and links. • Bioinformatics was born – a high-tech way to collate and access information from genetic databases.

Discussing the implications of the HGP Scrubs – Huntington’s Disease If you knew that a member of your family had a rare genetic disorder and you could be tested for it quickly and easily, would you do it? Why? Human Genome Project - Ethical, Legal, & Social Implications If you were invited to share your genome with researchers in the hope of finding cures for genetically-based illnesses, would you do it? Why? Robert Cook-Deegan on Patenting Genes Do you feel that gene patenting should be allowed? Why?

4.4.7When genes are transferred between species, the amino acid sequence of polypeptides translated from them is unchanged because the genetic code is universal All living things use the same bases (G.A.T.C!) A particular codon will produce the same amino acid, regardless of the species. This means the sequence of amino acids in a polypeptide remains unchanged. This allows us to take a gene from one species and insert it into the genome of another species. A well-known example of this gene transfer process is the production of human insulin by GM E. coli bacteria. A potential treatment for haemophilia is the injection of human clotting factors produced in the milk of GM sheep.

4.4.8A basic technique for gene transfer involves plasmids, a host cell, restriction enzymes and DNA ligase.

Recognition Site cut Recognition Site The restriction enzyme EcoRI cuts here cut cut RestrictionEnzymes • Naturally occurring bacterialenzymes are used as “molecularscalpels” • Allow genetic engineers to cut up DNA in a controlled way. • Restrictionenzymes are used to cut DNA molecules at very precise sequences of 4 to 8 base pairs called recognition sites. GAATTC GAATTC DNA CTTAAG CTTAAG

A restriction enzyme cuts the double-stranded DNA molecule at its specific recognition site A A T T C G C T T A A G The cuts produce a DNA fragment with two “sticky” ends A A T T C A A T T C G G The two different fragments cut by the same restriction enzyme have identical sticky ends and are able to join together G G C T T A A C T T A A When two fragments of DNA cut by the same restriction enzyme come together, they can join by base-pairing Sticky Ends Restriction enzyme: EcoRI • It is possible to use restriction enzymes that cut leaving an overhang; a so-called “sticky end”. • DNA cut in such a way produces ends which may only be joined to othersticky ends with a complementary base sequence. • See steps 1-3 opposite: Fragment A A T T C G C T T A A G Restriction enzyme: EcoRI Sticky end DNA from another source

C C C C C C G G G G G G G G G C C C G G G G G G C C C C C C DNA G G G C C C C C C G G G G G G C C C C C C G G G G G G C C C cut cut C C C G G G C C C G G G Blunt Ends Recognition Site Recognition Site • It is possible to use restriction enzymes that cut leaving no overhang; a so-called “blunt end”. • DNA cut in such a way is able to be joined to any other blunt end fragment, but tends to be non-specific because there are no sticky ends as recognition sites. Restriction enzyme cuts here The cut by this type of restriction enzyme leaves no overhang DNA from another source A special group of enzymes can join the pieces together

Two pieces of DNA are cut using the same restriction enzyme. The two different DNA fragments are attracted to each other by weak hydrogen bonds. This other end of the foreign DNA is attracted to the remaining sticky end of the plasmid. A A T T C G Plasmid DNA fragment Foreign DNA fragment G C T T A A Ligation • DNA fragments produced using restriction enzymes may be reassembled by a process called ligation. • Pieces of DNA are joined together using an enzyme called DNA ligase. • DNA of different origins produced in this way is called recombinant DNA because it is DNA that has been recombined from different sources. • Steps 1-3 are involved in creating a recombinant DNA plasmid:

Detail of Restriction Site Restriction sites on the fragments are attracted by base pairing only Gap in DNA molecule’s ‘backbone’ A T T A C G T A A T G C Plasmid DNA fragment Foreign DNA fragment C G T A A T G C T A A T Annealing • When the two matching “sticky ends” come together, they join by base pairing. This process is called annealing. • This can allow DNA fragments from a different source, perhaps a plasmid, to be joined to the DNA fragment. • The joined fragments will usually form either a linear molecule or a circular one, as shown here for a plasmid.

Detail of Restriction Site A T T A C G A T A T G C Recombinant Plasmid DNA Fragments linked permanently by DNA ligase No break in DNA molecule C G T A A T G C T A A T Recombinant DNA Plasmid • The fragments of DNA are joined together by the enzyme DNA ligase, producing a molecule of recombinant DNA. • These combined techniques of using restriction enzymes and ligation are the basic tools of geneticengineering. DNA ligase The fragments are able to join together under the influence ofDNA ligase.

Gene therapy using gene transfer A virus vector is used to insert the recombinant plasmid into the genes of affected cells. The virus is chosen or designed to target only those specific cells. Severe Combined Immune Deficiency can be treated this way Gene therapy ‘reverses’ hereditary blindness

4.4.9Give examples of the current uses of genetically modified crops or animals GMOs are already in circulation and have been produced for many uses, including agricultural and medical.

GM food and you 4.4.10 Discuss the potential benefits and possible harmful effects of one example of GM The ethical debate over GMOs rages on, and as scientists we must always bear in mind the precautionary principle: “If an action is potentially harmful, the burden of proof of safety lies with those who propose to take the action.”

4.4.11 Define clone A group of genetically identical organisms or groups of cells derived from a single parent cell Monozygotic (one zygote) twins are naturally occuring clones. Why do they not look identical?... Epigenetics has the answer Asexual reproduction (i.e. in bacteria) is an example of cloning Cloning via binary fission Taking plant cuttings and growing a new plant is also cloning. As is plants producing bulbs and runners

4.4.12 Outline a technique for cloning using differentiated animal cells Cloning cell cultures using nuclear transfer: Remove a differentiated diploid nucleus from the individual to be cloned Enucleate a donor egg cell Insert the diploid nucleus into the enucleated egg cell Stimulate it to divide and grow Collect cells for therapeutic purposes, such as creating skin tissue for burn patients Reproductive cloning using nuclear transfer: Remove the differentiated diploid nucleus from the individual to be cloned Enucleate the donor egg cell Insert the diploid nucleus into the enucleated egg cell Insert into a surrogate mother and gestate The newborn will be genetically identical to the donor nucleus parent

Reproductive vs therapeutic cloning • Reproductive cloning • The first mammal cloned was Dolly the sheep • Human cloning is illegal around the world • There will be no: “Mini-me” as in ‘Austin Powers’ (a clone to take over the world) or the characters played by Ewen McGregor and Scarlett Johansen in ‘The Island’ (spare parts “just in case”). • Therapeutic cloning is less controversial • It has the potential to treat (using stem cells) degenerative diseases like Parkinson’s disease and Multiple Sclerosis. • Does involve producing an embryo that could grow to full term (same as an IVF embryo) – but then it would be cloning! Dolly and her birth mom. A black faced ewe cannot have a white faced baby – so they must not be genetically related.

Critical considerations In 2008, a team led by DR Viviane Tabar extracted skin cells from the tails of mice with Parkinson’s disease. They removed the nucleus from each cell and implanted them into egg cells from which the nuclei had been removed. The resulting cells, which were genetically identical to the donor mice, developed as embryos and produced stem cells that could differentiate into dopamine neurons – the type that are missing in Parkinson’s disease. The team injected these stem cells into the affected regions of the brains of the donor mice and found that there was a marked improvement in the symptoms of Parkinson’s disease. Is it acceptable to create embryos as a source of stem cells for treatments that reduce suffering in a child or adult, but result in the death of the embryo?

The process of cloning Nucleus is removed ? Nucleus is removed

4.4.13 Discuss the ethical issues of therapeutic cloning in humans

Genetic engineering and biotechnology