Genetics & Evolution Series: Set 9 Version: 2.0

What is Gene Technology? Gene technology is a broad field which includes analysis of DNA as well as genetic engineering and other forms of genetic modification. Genetic engineering refers the artificial manipulation of genes: adding or subtracting genes, or changing the way genes work. Organisms with artificially altered DNA are referred to as genetically modified organisms (GMOs). Gene technologies have great potential to benefit humanity through: increasing crop production increasing livestock production preventing and fighting disease reducing pollution and waste producing new products detecting and preventing crime

Why Gene Technology? Despite potential benefits, gene technology is highly controversial. Some people feel very strongly that safety concerns associated with the technology have not been adequately addressed. Environmentally friendly Could improve the sustainability of crop and livestock production Could potentially benefit the health of many More predictable and directed than selective breeding Who owns and regulates the GMOs? Third world economies are at risk of exploitation Biological risks have not been adequately addressed Animal ethics issues The costs of errors Photos courtesy of GreenPeace

The Beginning of GE Genetic engineering (GE) was made possible by the discovery of new techniques and tools in the 1970s and 1980s. It builds on traditional methods of genetic manipulation, including selective breeding programs and the deliberate introduction of novel traits by exposing organisms (particularly plants) to mutagens. Methods were developed to insert ‘foreign’ DNA into cells using vectors. New recombinant DNA technology involved ‘recombining’ DNA from different individuals and even different species. Organisms such as bacteria, viruses, and yeasts are used to propagate recombinant genes and/or transfer genes to target cells (cells that receive the new DNA). The bacterium Escherichia coli (above) and the yeast Saccharomyces cerevisiae (below): favorite organisms of gene research

Producing GMOs GMOs may be created by modifying their DNA in one of three ways: Adding a Foreign Gene A foreign gene is added which will enable the GMO to carry out a new genetic program. Organisms altered in this way are referred to as transgenic. Host DNA Alter an Existing Gene An existing gene already present in the organism may be altered to make it express at a higher level (e.g. growth hormone) or in a different way (in tissue that would not normally express it). This method is also used for gene therapy. Existing gene altered Host DNA Delete or ‘Turn Off’ a Gene An existing gene may be deleted or deactivated to prevent the expression of a trait (e.g. the deactivation of the ripening gene in tomatoes). Host DNA

Restriction Enzymes Restriction enzymes are one of the essential tools of genetic engineering. Purified forms of these naturally occurring bacterial enzymes are used as “molecular scalpels”, allowing genetic engineers to cut up DNA in a controlled way. Restriction enzymes are used to cut DNA molecules at very precise sequences of 4 to 8 base pairs called recognition sites (see below). By using a ‘tool kit’ of over 400 restriction enzymes recognizing about 100 recognition sites, genetic engineers are able to isolate and sequence DNA, and manipulate individual genes derived from any type of organism. Recognition Site cut The restriction enzyme EcoRI cuts here GAATTC GAATTC DNA CTTAAG CTTAAG

Specific Recognition Sites Restriction enzymes are named according to the bacterial species they were first isolated from, followed by a number to distinguish different enzymes isolated from the same organism. e.g. BamHI was isolated from the bacteria Bacillus amyloliquefaciens strain H. A restriction enzyme cuts the double-stranded DNA molecule at its specific recognition site: Enzyme Source Recognition Sites EcoRI Escherichia coli RY13 GAATTC BamHI Bacillus amyloliquefaciens H GGATCC HaeIII Haemophilus aegyptius GGCC HindIII Haemophilus influenzae Rd AAGCTT Hpal Haemophilus parainfluenzae GTTAAC HpaII CCGG MboI Moraxella bovis GATC NotI Norcardia otitidis-caviarum GCGGCCGC TaqI Thermus aquaticus TCGA

DNA from another source Sticky Ends A restriction enzyme cuts the double-stranded DNA molecule at its specific recognition site 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 other sticky ends with a complementary base sequence. See steps 1-3 opposite: Restriction enzyme: EcoRI Fragment A A T T C C T T A A G G A A T T C C T T A A G Restriction enzyme: EcoRI Sticky end The two different fragments cut by the same restriction enzyme have identical sticky ends and are able to join together The cuts produce a DNA fragment with two “sticky” ends C T T A A A A T T C G C T T A A A A T T C G DNA from another source When two fragments of DNA cut by the same restriction enzyme come together, they can join by base-pairing

Restriction enzyme cuts here 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. C C C G G G C C C G G G G G G C C C G G G C C C DNA cut cut Restriction enzyme cuts here The cut by this type of restriction enzyme leaves no overhang C C C G G G G G G C C C G G G C C C DNA from another source A special group of enzymes can join the pieces together

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: Plasmid DNA fragment Two pieces of DNA are cut using the same restriction enzyme. This other end of the foreign DNA is attracted to the remaining sticky end of the plasmid. Foreign DNA fragment A A T T C G C T T A A The two different DNA fragments are attracted to each other by weak hydrogen bonds.

Detail of Restriction Site 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. Foreign DNA fragment A T C G Detail of Restriction Site Restriction sites on the fragments are attracted by base pairing only Gap in DNA molecule’s ‘backbone’ C A Plasmid DNA fragment G 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 genetic engineering. DNA ligase G A T C Recombinant Plasmid DNA Detail of Restriction Site Fragments linked permanently by DNA ligase No break in DNA molecule The fragments are able to join together under the influence of DNA ligase.

DNA Amplification 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 Samples from some sources, including those shown here, may be difficult to obtain in any quantity. A crime scene (body tissue samples) A single viral particle (from an infection) Fragments of DNA from a long extinct animal

PCR Equipment Amplification of DNA can be carried out with simple-to-use PCR machines called thermal cyclers (shown below). Thermal cyclers are in common use in the biology departments of universities as well as other kinds of research and analytical laboratories.

Steps in the PCR Process Separate Strands Separate the target DNA strands by heating at 98°C for 5 minutes The laboratory process called the polymerase chain reaction or PCR involves the following steps 1-3 each cycle: Repeat for about 25 cycles Repeat cycle of heating and cooling until enough copies of the target DNA have been produced. 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. Incubate Cool to 60°C and incubate for a few minutes. During this time, primers attach to single-stranded DNA. DNA polymerase synthesizes complementary strands.

Polymerase Chain Reaction PCR cycles No. of target DNA strands 1 2 4 3 8 16 5 32 6 64 7 128 256 9 512 10 1024 11 2048 12 4096 13 8192 14 16 384 15 32 768 65 536 17 131 072 18 262 144 19 524 288 20 1 048 576 21 2 097 152 22 4 194 304 23 8 388 608 24 16 777 216 25 33 554 432 Original DNASample Although only three cycles of replication are shown here, following cycles replicate DNA at an exponential rate 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

The Process of PCR 1 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

The Process of PCR 2 Nucleotides 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 synthesizes a complementary strand of DNA using free nucleotides. Nucleotides After one cycle, there are now two copies of the original sample.

Gel Electrophoresis Wells into which samples to be analyzed are placed. Buffer solution Cathode Anode Gel Plastic Frame Buffer Sample DNA fragments, shown symbolically above, move towards the positive terminal (smaller fragments move faster than longer ones). A technique known as gel electrophoresis can be used to separate large molecules (including nucleic acids or proteins) on the basis of their size, electric charge, and other physical properties. To prepare DNA for electrophoresis, the DNA is often cut up into smaller pieces. Called a restriction digest, and it produces a range of DNA of different lengths. To carry out electrophoresis, the DNA samples are placed in wells and covered with a buffer solution that gradually dissolves them into solution.

Tray: Contains the set gel. Analyzing DNA By applying an electric field to the solution, the molecules move towards one or other electrode depending on the charge on the molecule itself. DNA is negatively charged because the phosphates have a negative charge. Molecules of different sizes (molecular weights) become separated (spread out) on the gel surface. These can be visualized by applying dyes or radio-labeled probes. Tray: Contains the set gel. 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 seive for the DNA molecules. Wells: Holes created in the gel with a comb. -ve terminal +ve terminal Small fragments Large fragments

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 DNA restriction fragments or PCR products which is used to identify an individual. There are different methods of DNA profiling, each with benefits and drawbacks. 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.

Visualizing the Profile DNA fragments (PCR product after endonuclease digestion) visualized under UV light after staining with ethidium bromide and migration in an agarose electrophoresis gel.

DNA Profiling Methods DNA profiling begins by extracting DNA from the cells in a sample of blood, saliva, semen, or other fluid or tissue. Two methods are commonly used. Both are based on the analysis of short repetitive sequences in the DNA. Profiling using probes (RFLP analysis) was the first profiling technique to be developed. Restriction enzymes are applied to a DNA sample and the DNA fragments are separated on a gel. Radioactive probes are used to label DNA fragments with complementary sequences. Profiling using PCR is newer technique which uses highly polymorphic regions of DNA that have short repeated sequences of DNA. These sequences are amplified using PCR and then separated on a gel. This technique is suitable when there is very little DNA available or the sample is old.