Microbiology 2230 3/22 and 3/24 lectures
Microbiology 2230 3/22 and 3/24 lectures BIOL 2230
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This 5 page Class Notes was uploaded by Allison Collins on Thursday March 24, 2016. The Class Notes belongs to BIOL 2230 at Middle Tennessee State University taught by Anthony L Newsome in Fall 2015. Since its upload, it has received 61 views. For similar materials see Microbiology in Biology at Middle Tennessee State University.
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Date Created: 03/24/16
3/22 Genetic Probe • Probes can be made to identify a bacterium or a genetic disease • Ex: Angelina Jolie got a double mastectomy and hysterectomy because of a gene sequence found by a genetic probe that suggested a high probability of breast and cervical cancer • Probe: a DNA or RNA molecule which is used to locate a complementary RNA or DNA by hybridizing (through complementary base pairing) with it • Hybrid – double stranded nucleic acid in which strands differ in origin • ATCG sequence distinguishes between bacteria (and eukaryotes) • Manufacture complementary sequence (as a guess) and if it binds to extracted DNA on a slide, the particular ATCG sequence is present • Indicator molecule (color) – color change on slide indicates that DNA sequence stuck to slide, so its complementary strand is present DNA synthesis or oligonucleotides synthesis (in a lab) • About 2-‐30 bases long • 2 purposes o Probes à DNA hybridization à identify genes or bacteria o Primers à PCR à manufacture copies of a segment of DNA In situ hybridization – using genetic probe (oligonucleotides), indicator molecule • Cheaper than using agar plate; common process • Genetic sequences exist that are associated with any physical condition o Vary in levels of directness of relationship with condition Genetic ID card – 23 & Me • Cheap, accessible • Indicates the increased risk of diseases, not definite occurrence • Drop DNA strand onto silicon chip and test for attachment to genetic probe • FDA pulled most advertising to avoid mass panic/misunderstanding by the public, but it is still available at medical centers • Ethical questions o Do insurance companies have the right to know your test results? o Results on a developing fetus could affect parents ’ decision to follow through with pregnancy or more testing • Can also associate sequences with mental issues such as bipolar disorder, schizophrenia, depression, personality traits o Does an employer have the right to know? • Employers have been found to secretly test blood samples in some cases • Horse racing – “speed gene” test Babies – triple screen test • High percentage of false positive results • Genetic counselors can help analyze results • FISH – fluorescent in situ hybridization o Amniocentesis – extract amniotic fluid – risky procedure o Double check triple screen for accuracy • High probability that you’ll be genetically probed at some point in your lifetime Polymerase Chain Reaction • Kary Mullins developed it and won Nobel Prize in 1993 • Jurassic Park novel based on PCR 2 3/24 Polymerase Chain Reaction • Relatively simple process PCR definition: an in vitro (in a test tube) reaction in which a specific region of DNA is amplified many times by repeated synthesis of DNA using DNA polymerase and specific primers to define the ends of the amplified region • Application: make large quantities of a particular DNA sequence • Primer – a piece of DNA that provides an end to which DNA polymerase can add nucleotides – approx. nucleotides long in this process Fundamental steps • 1. Synthesize fragments with sequences (primers) identical to those on either side of the targeted sequence (approx. 20 nucleotides) • 2. Denature DNA by heating it to 94C for 15 seconds o Separate 2 DNA strands • 3. Add multiple primers and lower temperature to 68C for 60 seconds to allow primers to anneal (hydrogen bond) to DNA o Because the primers are added in excess, the the targeted DNA strands will almost always anneal to the primers rather than to each other • 4. Add nucleotide triphosphates and DNA polymerase • 5. The DNA polymerase extends the primers and synthesizes copies of target DNA sequence o 2 strands à 4 strands • 6. Repeat heating and cooling cycle and each cycle generates a complementary strand from each preexisting strand o 20 cycles will produce about 1 million copies o Pieces ranging in size from <100 base pairs to several thousand base pairs in length can be amplified 3 • Use a heat stable polymerase from a thermophilic bacteria – only polymerases are able to function at the high temps used in the PCR o Taq polymerase -‐ from Thermus aquaticus, bacteria from hot springs o Vent polymerase – from Thermococcus litoralis, bacteria from deep sea vents • Forensics application: rape case in Chattanooga o Semen from rape victims was used to generate RFLP from DNA o Cigarette butt from rape suspect used to extract small amount of DNA and amplify with PCR o DNA from semen matched with DNA from cigarette butt, suspect convicted o Has since been deemed unlawful to confiscate DNA in such a manner unless suspect is a convicted felon Recombinant DNA technology (rDNA) • Hepatitis vaccine – originally very expensive, today is free o Original method: extract and kill virus from blood sample from someone who is Hepatitis positive o Now: take nucleic acid from virus, introduce to bacteria, bacteria produces proteins à use protein for vaccine rDNA technology in bacteria based on: • 1. Restriction endonucleases o found in bacteria – cut DNA (not found in humans) • 2. DNA ligases (join ends of DNA) • 3. Plasmids • 4. Gram negative bacteria E. coli is most commonly used bacteria in biotechnology (most strands are nonpathenogenic) 4 • Easy to grow • Dirt cheap to grow • Grows quickly • We know its genetics well – easy to place genes into it that are responsible for protein production Bacterial production of proteins used for vaccines • Take cells producing desired product (eukaryotic or prokaryotic) o Isolate DNA and treat with restriction endonucleases o Some of the DNA fragments have desired genes • Take bacteria resistant to ampicillin (antibiotic) and resistance carried on plasmids o Isolate plasmids and break open with restriction enzymes • Mix DNA fragments from cells with desired product + broken plasmids from abx-‐resistant bacteria • Add DNA ligase to join ends together o Now you have plasmids with antibiotic genes and foreign DNA • Screen bacteria for production of desired protein and product o Add bacteria (E. coli) sensistive to ampicillin (i.e. don’t have plasmids) to agar plate containing ampicillin § Often plasmids don’t get into bacteria – check for presence on an agar plate § Only E. coli with plasmids (recombinants) will grow § Test each colony on the agar plate for proteins § One colony with the protein means success – can now duplicate bacteria from that colony 5
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