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PCR方法检测Alu重复序列

发布时间:2019-08-26 07:15编辑:生命科学浏览(138)

    核心提示:collect piece of tissue in Eppendorf tube containing 40 ml 0.25 N NaOH put in

    核心提示:collect piece of tissue in Eppendorf tube containing 40 ml 0.25 N NaOH put in

    核心提示:Purpose: Mu TAIL PCR produces a population of fragments with one end anchored in Robertson's Mutator TIR's a

    核心提示:In this experiment, polymerase chain reaction is used to amplify a nucleotide sequence from chromosome 8 to look In this experiment, polymerase chain reaction is used to amplify a nucleotide sequence from chromosome 8 to look for an insertion of a short DNA sequence called Alu within the tissue plasminogen activator gene. Although the DNA from different individuals is more alike than different, there are many regions of the human chromosomes that exhibit a great deal of diversity. Such variable sequences are termed "polymorphic" and provide the basis for genetic disease diagnosis, forensic identification, and paternity testing.

    1. collect piece of tissue in Eppendorf tube containing 40 ml 0.25 N NaOH
    2. put in boiling H2O for 30 sec
    3. neutralize by adding 40 ml 0.25 N HCl and 20 ml 0.5 M Tris-HCl pH 8.0, 0.25% Igepal CA-630
    4. boil for another 2 min
    5. use directly for PCR or store at 4°C for up to several weeks
    6. if stored at 4°C boil again for 2 min before the PCR
    1. collect piece of tissue in Eppendorf tube containing 40 ml 0.25 N NaOH
    2. put in boiling H2O for 30 sec
    3. neutralize by adding 40 ml 0.25 N HCl and 20 ml 0.5 M Tris-HCl pH 8.0, 0.25% Igepal CA-630
    4. boil for another 2 min
    5. use directly for PCR or store at 4°C for up to several weeks
    6. if stored at 4°C boil again for 2 min before the PCR

    Purpose: Mu TAIL PCR produces a population of fragments with one end anchored in Robertson's Mutator TIR's and the other in the Mu flanking DNA. These products may be used for cloning, gel analysis, or as hybridization probes. This protocol details the steps to take to improve the chances for uncontaminated and reproducible TAIL reactions. The protocol is adapted from the published method .Template preparation: Fast Isolation of Genomic DNAThis protocol is used for quick isolation of genomic DNA from plant materials. Other methods may also prove effective.DNA extraction buffer: Store at RT.

    The Alu family of short interspersed repeated DNA elements are distributed throughout primate genomes. Over the past 65 million years, the Alu sequence has amplified via an RNA-mediated transposition process to a copy number of about 500,000--comprising an estimated 5% of the human genome. Alu sequences are thought to be derived from the 7SL RNA gene which encodes the RNA component of the signal recognition particle that functions in protein synthesis. Alu elements are approximately 300-bp in length and derive their name from a single recognition site for the endonuclease Alu 1 located near the middle of the Alu sequence.

    Remarks:

    We don't have too much experience with this method. Mostly it seems to work though. However, I don't think it is safe enough for critical purposes .

    Remarks:

    We don't have too much experience with this method. Mostly it seems to work though. However, I don't think it is safe enough for critical purposes .

    168 g Urea25 ml 5 M NaCl20 ml 1 M Tris-HCl pH 8.016 ml 0.5 M EDTA20 ml 20% Sarkosyl190 ml distilled H2O1.

    An estimated 500-2,000 Alu elements are mostly restricted to the human genome. A few of these have inserted recently, within the last one million years, and are not fixed in the human species. One such Alu element, called TPA-25, is found within an intron of the tissue plasminogen activator gene. This insertion is dimorphic, meaning that it is present in some individuals and not in others. PCR can be used to screen individuals for the presence of the TPA-25 insertion.

    Materials:

    Reagent

    Supplier

    Cat.-#

    澳门金莎娱乐网址 1

    Grind about 1 g of fresh seedling leaves, or other tissues, to a fine powder in liquid nitrogen using a mortar and pestle. Use more tissue from other plant parts or older plants. Be careful to avoid contaminating adjacent samples with the powder.

    In this experiment, oligonucleotide primers, flanking the insertion site, are used to amplify a 400-bp fragment when TPA-25 is present and a 100-bp fragment when it is absent. Each of the three possible genotypes--homozygotes for presence of TPA-25 , homozygous for absence of TPA-25 , and heterozygotes are distinguished following electrophoresis in agarose gels.

    1. Add 5 ml DNA extraction buffer. Mix gently with the pestle to evenly distribute the powdered tissue in the buffer.

    2. Let it thaw to room temperature and mix gently with the pestle again.

    3. 澳门金莎娱乐网址,Transfer to a polypropylene centrifuge tube .

    4. Add 4 ml TE8--saturated phenol:chloroform . Close cap. Shake briefly with hands and put the tubes, laying horizontally in a tightly closed container, on the belly dancer or similar gyrating platform mixer at the highest speed for at least 30 min. Wear gloves and goggles when handling phenol:chloroform.

    5. Spin at ~1625g for 5-20 mins . There will be a solid middle phase that separates the organic bottom layer from the aqueous top layer.

    6. Pour the upper layer into a fresh centrifuge tube . You may have to use a pipette if the middle pellet is not thick enough. The aqueous volume should be about 5 ml.

    7. To the supernatant, add 0.5 ml 3 M NaOAc . Mix gently by inverting the capped tube several times and a color change will occur.

    8. Add 3.9 ml isopropanol. Mix gently by inversion. In most cases, a clump of thread-like DNA is visible.

    9. Either use a glass pipette hook to fish out the DNA if it is visible , or centrifuge at 10,000 rpm for 10 min to pellet the DNA.

    10. Wash the DNA clump by transferring it to a microcentrifuge tube containing 1 ml 70% EtOH. Centrifuge at 10,000 rpm for 5 min at 4°C.

    11. Drain the EtOH by carefully inverting the tube on a KimWipe for a few minutes, then dry the DNA in a vacuum dryer for about 10 min. Do not overdry as it will become nearly insoluble.

    12. Add 200 – 400 ml TE8 buffer to dissolve the DNA. Leave it at 4 °C a few hours or overnight. The concentration should be approximately 100 ng/μl. Run 1 μl with and without DNase-free RNase on a 0.8% gel to check the prep for quality and concentration.

    13. To minimize freeze-thaw cycles, make 3 μl aliquots of each DNA in PCR tubes and store at -20°C. Multiple DNA samples can be arrayed in strip tubes for convenient dispensing into the reaction later. Use the aliquots within a few months.

    14. To use, make a 1:20 dilution by adding 57 μl of HPLC quality water to each aliquot. In some cases, this dilution should be adjusted if the DNA prep is at a concentration much different from 0.1mg/ml. Keep on ice. Discard after one use—do not store the dilution nor refreeze the aliquot.

    The source of template DNA is a sample of several thousand cells obtained by saline mouthwash . The cells are collected by centrifugation and resuspended in a solution containing the resin "Chelex," which binds metal ions that inhibit the PCR reaction. The cells are lyzed by boiling and centrifuged to remove cell debris. A sample of the supernatant containing genomic DNA is mixed with Taq polymerase, olignucleotide primers, the four deoxyynucleotides, and the cofactor magnesium chloride. Temperature cycling is used to denature the target DNA, anneal the primers, and extend a complementary DNA strand. The "upstream" primer, "5-GTAAGAGTTCCGTAACAGGACAGCT-3", brackets one side of the TPA locus, while the "downstream" primer, "5-CCCCACCCTAGGAGAACTTCTCTTT-3" brackets the other side. The size of the amplification product depends on the presence or absence of the Alu insertion at the TPA-25 locus on each copy of chromosome 8.

    Lab bench hygiene: contamination prevention, done for each experiment

    In order to compare the genotypes from a number of different individuals, aliquots of the amplified sample and those of other experimenters are loaded into wells of an agarose gel--along with the DNA size markers and an unamplified control. Following electrophoresis and staining, amplification products appear as distinct bands in the gel--the distance moved from the well is inversely proportional to the presence or absence of the TPA-25 insertion. One or two bands are visible in each lane, indicating that an individual is either homozygous or heterozygous for the Alu insertion.

    1. Wash the lab bench with a mixture of bleach, detergent and water.

    2. Wipe down freezer and refrigerator handles with dilute bleach.

    3. Wash the boxes you will use to hold the PCR plates and tubes with dilute bleach.

    4. Clean your pipettors with 70% EtOH and compressed air . It is best to have a set dedicated to PCR that are always used with filter tips. Wipe them down again after pipetting template solutions.

    5. Never touch anything that you use for TAIL reactions unless you have clean gloves on. Store all disposables in a dedicated drawer that you open only with clean gloves on.

    6. Keep lids closed on pipette tip boxes and reagents. Do not use PCR reagents for other purpos

    7. Use aerosol-resistant tips for all pipetting steps.

    8. Keep everything at 4°C and covered while reactions are set up. I recommend using Eppendorf PCR Coolers which stay at this temperature for 1 hour. As an alternative, some types of pipette tip boxes can be used as ice chests to conveniently hold PCR tubes and plates.

    9. Change gloves whenever you leave your bench area or if you handle a DNA solution. Discarded gloves may be re-used for other activities for which contamination is not an issue.

    Reagents10% Chelex 0.9% sodium chloride PCR reaction mix 25 mM MgCl2 mineral oil DNA size markers distilled water loading dye 1.5% agarose 1X Tris/Borate/EDTA buffer 1 micrograms/ml ethidium bromide For Decontamination:0.05 M KMnO4 0.25 N HCl 0.25 N NaOH

    Equipment and Supplies100-1000 microliter micropipettor tips 10-100 microliter micropipettor tips 0.5-10 microliter micropipettor tips 15 ml culture tube 1.5 ml tubes 0.5 ml PCR tubes beaker for waste/used tips boiling water bath DNA thermal cycler forceps paper cup Procedure Use permanent marker to label your name on a 15-ml culture tube containing saline solution.

    Pour all of the saline solution into your mouth, and vigorously swish for 10 seconds. Save the empty 15-ml tube for reuse in the next step.

    Expel saline mouthwash into a paper cup. Then carefully pour saline mouthwash from paper cup back into 15-ml tube from Step 1.

    Securely close cap, and place mouthwash tube in a balanced configuration with other tubes in rotor of clinical centrifuge. Centrifuge at 500-1,000 x g for 10 minutes to pellet cells on the bottom side of culture tube.

    Being careful not to disturb cell pellet, pour off as much supernatant as possible into sink or paper cup. Place tube with mouthwash cell pellet on ice.

    Use micropipettor to add 500 microliters of 10% Chelex to cell pellet:

    a. Pipet Chelex solution in and out of pipet tip several times to suspend the Chelex beads.

    b. Before the Chelex has a chance to settle, transfer the 500 microliters to the culture tube.

    Resuspend cells in the Chelex by pipetting up and down several times. Hold tube up to light to confirm that no visible clumps of cells remain.

    Transfer 500 microliters of the resuspended mouthwash sample into a clean 1.5 ml reaction tube labeled with your name.

    Incubate the 1.5 ml sample tube in a boiling water bath for 10 minutes.

    Following incubation, use forceps to remove sample tube from boiling water bath, and cool tube on ice for approximately one minute.

    Place sample tube in a balanced configuration in a microfuge rotor, and spin for 30 seconds to pellet Chelex beads at bottom of tube.

    Transfer 200 microliters of the supernatant to a fresh 1.5 ml tube labeled with your name, and place tube on ice. Avoid transferring any of the Chelex pellet.

    Set up PCR reaction using 5 microliters of DNA sample in a 50 microliter reaction volume.

    Amplify using the following program:

    94 degrees C for 1 minute

    58 degrees C for 2 minutes

    72 degrees C for 2 minutes 30 cycles

    Electrophorese 10 microliters of the PCR reaction in a 1.5% agarose gel, stain, view, and photograph.

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