Miscellaneous from molecular techniques given in the first class
Miscellaneous from molecular techniques given in the first class BCH 6713
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This 9 page Class Notes was uploaded by Marina Notetaker on Tuesday September 13, 2016. The Class Notes belongs to BCH 6713 at Mississippi State University taught by Zhaohua Peng in Fall 2016. Since its upload, it has received 6 views.
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Date Created: 09/13/16
SOME MOLECULAR TECHNIQUES Note: it does not include all methods discussed in the slides! 1) Restriction enzymes main ideas: - it was first described in bacteria that use these enzymes to protect themselves against foreign DNA that tries to invade the cell, such as viruses. - restriction enzymes are enzymes that cut the DNA backbone on both sides, originating a break on the DNA sequence. It targets specific palindrome sequences, which are short sequences that can be read back and forward as same sequence. For example: AGCGA. - resulting cut fragments can be classified as (1) blunt ended strands – which are symmetric cut of the DNA or (2) sticky ended strands – which are asymmetric and can be (2a) 3’ sticky end cut, in which the overhanging part is at the 5’ and at the 3’ we have the smallest cut piece (2b) 5’ stickyend cut that has the 3’ overhanging strand. - bacteria protect itself from restriction enzyme by having a non-methylated DNA. 2) The main principle of molecular cloning is getting two DNAs, cutting them with same restriction enzyme (RE) that produce sticky ends and putting them together so they will recombine and form a single DNA sequence. This procedure often involves E.coli or other bacteria, plasmids, a target (insert) DNA and a RE that produce sticky ends, such as EcoR1 or HindIII. Events: two tubes, one with plasmids and other with target DNA > RE added to both tubes > mix the open plasmid with the small strand of DNA target >bycomplementarybases onthestickyends insertion occurs > plasmids are inserted on bacteria by either electroporation or the bacteria take up by themselves > growth bacterial colonies > genetic markers can be used to select only the bacteria that presents the recombined plasmid. 3) The most common way of selection of bacterial colony is by α-complementation. In this method the intact plasmid had an intact LacZ gene. The inserted foreign DNA breaks down the LacZ gene, so the bacteria with recombined plasmid cannot efficiently metabolize the lactose. By using specific media for culturing the cells, recombined bacteria will have white color while not recombined bacteria (due to no absorption of plasmid or having an intact plasmid) will have blue color. So, white colonies are selected. 4) Constituents of a clone: - Insert: the target foreign DNA that will be inserted on the vector. Called DNA clones. - Vector: is a DNA sequence in which will receive the foreign DNA. Often is a plasmid, but viruses can also be used as same purposes. - Host cell: cell in which the vector will be transferred and will producecopiesofthetargetDNAoritsproduct (protein).Vector can be transferred to the host cell by using competent cells (absorption naturally) or doing electroporation (shock treatment on the cells that make their membrane permeable). 5) Advantages of inserting a clone into bacteria: a. low cost and fast method, easy to assay b. E. coli can divide once every 20 minutes in ideal conditions. At the end of 10h can have billions of bacteria. c. DNA clones will be amplified without using PCR methods. 6) Main conditions to E.coli to growth: enough space on a plaque, no competition, fresh media and 37°C. 7) Plasmid is an extrachromosomal DNA. Often circular and double stranded, that can replicate itself whenever it wants. It does not require the cell to be on replication. Its main features include: a. Genetic Marker: it is a gene that allows the identification of the bacteria that had up taken the recombined plasmid, such as LacZ gene. It is related to the polycloning sites, because once an insert gets into the plasmid, it inactivates the marker causing changing on phenotype. b. Antibiotic resistant gene: gives advantage for growing bacteria, such as ampicillin resistance. You can also select bacteria by growing them in a media with antibiotic, so you know that the ones that are replicating have the plasmid. c. Polycloning sites: region formed by short sequences in which RE will act and the DNA will be inserted, such as the β-galacto site. Only a specific region has these sites on the plasmid. d. Partitioning genes: form genes that clock the entrance of other plasmids inside the bacteria, so only one plasmid per bacterium is found. It is naturally found in bacteria. 8) Plasmid is not very often found in bacteria. Usually few bacteria have plasmids. 9) DNA cloning libraries are the DNA products from a cloning. They can be classified as: a. Expression clone libraries: in which the plasmids also contain genes that allow the transcription and translation of the insert DNA. Vector needs to have promoter and initiation regions. So proteins can be analyzed and kept. This one is pretty muchused forpharmacological purposes, suchas productionofhuman insulin. The gene responsible for producing insulin is cloned and the protein formed bybacteria. Before it was collected from pigs and would cause reactions on humans and take lots of pigs to obtain insulin. b. Genomic clone libraries: formed by the target gene that was inserted. c. cDNA clone libraries: formed by mRNA that were reverse transcribed to cDNA and inserted into a clone. 10)The main methods of picking up colonies of bacteria are (1) by hand using tooth pick or (2) robots (select round, white, lonely colonies). Procedures after collecting colonies include: - Transferring colony to a new 384 well plate - Overnight growing conditions (inoculation) - Identifying the plate (bar codes) - Replicating the plates (usually more 3 plates) - Refrigeration -80°C 11)Main methods of plasmid/ DNA isolation include: - Electrophoresis gel - Pulsed-field electrophoresis - Blotting techniques - PCR - FISH 1. Electrophoresis Gel: use a gel of agarose or acrylamide. These gels have porous that allows the DNA to pass through. The gel is placed on an electro field container that has two fields (+) and (-). Over the gel a solution buffer is placed. Because the DNA backbone is negativelycharged, it migrates on the gel to (+) cathode. So, DNA is placed on small wells close to the (-) anode and migrates all over the gel trying to get closer to the (+) cathode. Larger fragments of DNA run slowly through the gel, so often stay more close to the initial well. Smaller fragments run faster and so go farther away from the initial point. So rate of migration is inverse proportional from the size of the DNA. Advantage: it is relatively cheap (mainly the agarose), easy and fast. Used when the DNA fragments are not much large. 2. Pulsed-field electrophoresis: it works basically just like the first one. However, instead of 2 fields (+) and (-), it is a 6 sided field in which the gel is placed in the middle, also using a buffer. The idea is the with increasing fields larger fragments can run through the gel without getting stuck. Each well can have more than 100 fragments with thousands bp. 3. Blotting techniques: basically it is a post electrophoresis method to obtain DNA or bacterial colony. Once you have your gel with theseparatedDNAfragments, a nylon or nitrocellulose filter is placed over the gel and over the filter a type of absorbent sponge. Under the gel, the buffer solution is kept. So, the buffer runs into the absorbent sponge by capillarity, carrying together the DNA fragments. Due to the charge of the filter, DNA is retained there. Type of blotting techniques include: - Western blotting: used to separate proteins -Southern blotting: used to separate DNA -Northern blotting: used to separate RNA -Colony blotting: used to separate bacterial colonies. After getting the colonies on the nitrocellulose or nylon filter, 0.5N NaOH is used to break the membrane of the bacteria and turn dsDNA into ssDNA. Proteases and other buffers are used to only leave intact DNA. All of them you can use a final target labeled (probe) that will recognize specifically the DNA fragments you want and fluoresce when binding to it. 4. PCR methods: it is an exponential amplification of DNA fragment. basically you will have primers designed for the specific DNA fragment you want. Different thermal cycles will be performed so you can have amplification of your target DNA: - 95C: DNA denaturation, open the dsDNA in ssDNA - 55-60C: primer annealing - 72C: DNA polymerase (Taq poly) extend your DNA sequence As replication, it requires: (1) DNA polymerase (2) dNTPs (3) primer complementaryto the vector flanking region. Most used DNA polymerase is Taq polymerase, isolated from Thermus aquaticus, a thermophilic bacterium. So the polymerase is resistant to high temperatures. At the end of first cycle you have 2 copies of DNA from the first DNA. At the end of ‘n’ cycles you have 2 copies. Usually 30 cycles are performed in a PCR. Advantage: more than isolate, more sequences are obtained. Disadvantage: size. Really long sequences won’t work because polymerase might fall at some point. 5. FISH: fluorescent in situ hybridization. It is a means by which genes and/or repeat sequences are visualized in the chromosome. This method is used to help recognizing the DNA sequences that you want. Basically you have your DNA sequence or chromosome and you insert it in a glass. The glass is heat up and chemicals are added to denature the DNA and turn it into ssDNA. Complementary specific probes bond to a hapten recognize its specific region on DNA sequence and hybridizes. After hybridization, another molecule is added to the glass. This has a fluorescent characteristic and is highly specific to the hapten. Once this molecule binds to the hapten, light is produced and captured by the machine. - hapten is an inert small molecule that does not affect hybridization procedure or biological process, such as biotin. - fluorescent tag specific for the hapten can be either another molecule such as streptavidin (highly specific to biotin) or an antibody. Uses in studies of (1) genetic recombination (2) relationship between genes (3) chromosome structure.
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