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DNA & BIOTECHNOLOGY DNA & BIOTECHNOLOGY Keywords Keywords Biotechnology Recombinant DNA Gene splicing Vector Plasmid vector Phage vector Transgenesis Transgenes Cloning Polymerase chain reaction Primer Short tandem repeats Gel electrophoresis DNA blot / Southern blot Capillary electrophoresis DNA micro-array WHAT IS BIOTECHNOLOGY? Biotechnology is using living things to create products or to do tasks for human beings. It is the practice of using plants, animals and micro-organisms and their biological processes to some benefit eg. in medicine, agriculture and industry Researchers use DNA, genes, yeast, bacteria and cells WHY USE BIOTECHNOLOGY? For ourselves For the environment Biotechnological research has been used to assist human health in many areas: Biotechnology is a tool used: antibiotics vaccines IFV genetic disorders dna profiling & forensics to help control pests for conservation of plant & animal species leach metals from the soil for cleaner mining clean up heavy metal contamination WHY USE BIOTECHNOLOGY For food & agriculture Selective or conventional breeding of plants and animals changes the genetic make-up of organisms. Gene technology makes these changes more specific. Biotechnology is used to create: Frost, salt and drought tolerant plants Salad vegetables that do not ‘brown’ Fruits and vegetables with extra vitamins Slow-ripening tomatoes & pineapples Produce blowfly-resistant sheep Increase wool production For horticulture Long-lasting flowers Blue roses For research & medicine Transgenic mice Production of insulin Production of antibiotics BIOTECHNOLOGY TERMS Term Meaning Gel electrophoresis The use of an electric charge applied to a gel in order to separate fragments of DNA according to their size Gene splicing Inserting foreign DNA into the DNA of a cell Gene vector Mechanical or biological, used to transfer DNA into a host cell. Phage vector (viral) or plasmid vector (bacterial) Ligase An enzyme which can be used to join DNA strands or the ‘sticky’ ends of DNA strands. Polymerase Chain Reaction A means of producing large quantities of a small sample of DNA by heating and cooling it many times in the presence of a heat tolerant enzyme called polymerase. The polymerase enables free nucleotides to combine with the heat separated threads of DNA as they are cooled, therefore doubling the amount of DNA each cycle. Recombinant gene A gene which has been inserted on a foreign organism’s DNA RECOMBINANT DNA & GENE SPLICING Recombinant DNA is a method of cutting and pasting a foreign piece of DNA into the DNA of a cell. It brings together genetic material from multiple sources, creating new sequences of DNA. Enables the genome to be manipulated very precisely Transgenesis Transgenesis is when specific genes from one organism are placed in the DNA of another organism of a different species The recombinant genes are called transgenes Example: Genetically modified mice for the purposes of scientific studies TRANSGENESIS Another example of transgenesis is for the production of specific proteins The first chemical produced by transgenesis was human insulin in the late 1980’s The human gene is placed into a bacterium which can then use the genetic information to produce the human hormone. The hormone is refined from the culture of bacteria. RECOMBINANT DNA & GENE SPLICING Steps to gene splicing: 1. Restriction enzymes Cut the DNA at a specific location Leaves the DNA strand with ‘sticky ends’ 2. Sticky ends Unattached (unpaired) nucleotides Match up with the DNA to be inserted 3. Ligation Ligase enzymes help form the hydrogen bonds between nucleotides DNA-ase helps form the bonds between the side strands (backbone) Fluorescent green protein transgenic mouse RECOMBINANT DNA & GENE SPLICING RECOMBINANT DNA & GENE SPLICING Sticky ends BamHI: HindIII EcoRI Vectors are the carriers of the recombinant DNA They can be phage (viral DNA) or plasmid (bacterial DNA) vectors Plasmids are small sections of DNA separate to the chromosomal DNA Most often, plasmids are genetically engineered Many bacteria however, also contain plasmids naturally VECTORS AND TRANSGENESIS RECOMBINANT DNA & GENE SPLICING Sticky ends The vector used in this diagram is a plasmid vector Some strains of the bacterium E. coli have resistance to the antibiotic kanamycin, others have resistance to tetracycline. Plasmid* carrying gene for Plasmid* carrying gene for kanamycin resistance tetracycline resistance K E. coli with kanamycin resistance T E. coli with tetracycline resistance * Plasmids are small circular pieces of DNA that occur in bacteria and protozoa. They are not in a chromosome but can replicate independently in a host cell. K Plasmids can be cut at specific points using restriction enzymes T The cut plasmids are mixed with DNA ligase to form recombinant DNA K T Plasmid reintroduced into E. coli E. coli with tetracycline and kanamycin resistance POLYMERASE CHAIN REACTION The polymerase chain reaction (PCR) is a process that reproduces large quantities of small sections of DNA. Background information The process involves heating and cooling cycles. It has been made possible with the discovery of the Taq polymerase enzyme which is heat tolerant. This enzyme was originally discovered in a bacteria (Thermus aquaticus) that lives in hot springs It can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72°C. Young man with cystic fibrosis taking medication using a nebuliser. Wellcome Library The gene responsible for cystic fibrosis has been identified and it is hoped that, using recombinant DNA technology, it will be possible to transfer a normal copy of the gene into affected cells. GENE THERAPY It has long been anticipated that cystic fibrosis will be one of the first diseases to be treated by gene therapy. However, since the first clinical trials in the early 1990s numerous problems have been encountered. It is expected that a clinically effective treatment will be available in the next 10 years. POLYMERASE CHAIN REACTION Steps 1. The DNA to be copied is heated to 90ºC, which causes the strands to separate (denature). 2. Large amounts of primers are added to the single strands of DNA. 3. These primers bind to the matching DNA sequences in front of the gene that is to be copied. This tells the polymerase enzyme where to start copying. 4. The primers are both forward and reverse sections of DNA (5’ to 3’ and 3’ to 5’). 5. 6. 7. 8. 9. The mixture is then cooled so that the primers can anneal (bind) to the single stranded templates. The polymerase enzyme is added and the mixture is heated again to about 70ºC. DNA polymerase begins adding nucleotides onto the ends of the annealed primers. At the end of the cycle, which lasts about 5 minutes, the temperature is raised and the process begins again. The number of copies doubles after each cycle. Usually 25 to 30 cycles produce a sufficient amount of DNA. PCR MACHINE POLYMERASE CHAIN REACTION POLYMERASE CHAIN REACTION DNA SEQUENCING / PROFILING DNA sequencing is used to work out the exact order of the base pairs in a section of DNA. Knowing the base sequence can be helpful in locating and identifying specific genes. Gene probes can then be made and used to locate these genes DNA profiling is used to identify species or individuals Gel electrophoresis is used in DNA profiling. SHORT TANDEM REPEATS DNA profiling uses the "repeat" sequences of non-coding DNA that are found between the genes that code for proteins. These sequences can vary a great deal between individuals (polymorphic). They are called short tandem repeats (STRs). The number of repeats is inherited. Therefore, unrelated individuals are extremely unlikely to have the same number. Gel electrophoresis separates these fragments of DNA (STRs) using an electric current. DNA has a slight negative charge. It will migrate towards the positive end of the gel. Smaller fragments move faster through the gel than larger fragments. The gel can be upright like this one or it an be a flat bed gel – both work in the same way. At the end of the ‘run’ a pattern of bands will be produced. Each band represents a fragment size of DNA from the sample. Different samples will have different patterns. GEL ELECTROPHORESIS FRAGMENT LENGTHS AND GEL ELECTROPHORESIS SHORT TANDEM REPEATS Blotting is a method of ‘photographing’ the resulting sequence of DNA fragments once they have gone through the process of gel electrophoresis. DNA BLOT ANALYSIS In the example below, what is the genotype of the father? Rule out all the mother’s alleles. The ones left are from the father. DNA BLOT ANALYSIS Who was adopted? DNA PROFILING - SUMMARY Steps in DNA profiling 1. Collect sample of material containing cells 2. Extract DNA from sample 3. Use PCR to increase the size of the sample 4. Add restriction enzymes to DNA sample 5. Place solution of DNA and restriction enzymes into gel electrophoresis 6. Run gel 7. Process gel to see location of DNA bands 8. Photograph the gel (DNA or Southern blot) CAPILLARY ELECTROPHORESIS • STR profiles are more easily stored and compared in the form of numbers and letters rather than pictures of lines. • Capillary electrophoresis is a way of collecting numerical data that is plotted on a line graph. • The peaks on the graph represent the different STRs Sample Amelogenin D3S1358 vWA FGA D8S1179 D21S11 D18S51 Victim XY 14, 15 18, 20 24 13, 16 28, 30.2 14, 15 Suspect XY 14, 15 15, 18 21, 22 13, 14 30 14, 15 Blood Stain from Crime Scene XY 14, 15 15, 18 21, 22 13,14 30 14, 15 Capillary electrophoresis graph USES OF DNA PROFILING Identification Criminals Victims – crimes, disasters Family members Species – quarantine, smuggling Genetic differences between populations Information is kept in data banks GENE PROBES The search for a particular gene uses a single-stranded piece of DNA called a gene probe. To test a DNA sample, it is first treated so that the double-stranded molecule unzips into single strands. The probe is then added to the solution Probes are constructed with a radioactive or a fluorescent section (tag) to that they can be detected after attaching to the DNA. The probe gives infomration about which chromosome the gene is on, and where the gen is on the chromosome. We know the base sequences in a number of disease-causing genes. Gene probes can detect if these genes are present in individuals being tested. DNA MICRO-ARRAYS o A DNA mico-array allows scientists to perform an experiment on thousands of genes at the same time. o Each spot on a micro-array contains multiple identical strands of DNA. o The DNA sequence on each spot is unique. o Each spot represents one gene. Thousands of spots are arrayed in orderly rows and columns on a solid surface (usually glass). o The precise location and sequence of each spot is recorded in a computer database. o Microarrays can be the size of a microscope slide, or even smaller http://learn.genetics.utah.edu/content/labs/microarray/