Lab 3: amplification and isolation of enhancer using PCR & agarose gel extraction

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1 Lab 3: amplification and isolation of enhancer using PCR & Purpose The goal of this lab is to: 1) Dilute your lyophilized primer oligonucleotides to a suitable storage concentration. 2) Design and execute a PCR reaction to amplify the proximal promoter region of your assigned gene using zebrafish genomic DNA and the primers that you designed previous labs. 3) Characterize the amplicon product of the PCR reaction from lab by agarose gel electrophoresis. 4) Extract the specific amplicon from the agarose gel using a modified Vogelstein and Gillespie extraction 1. Assignment Summary Maintain careful notes reflecting modification, deviation, or elaboration of the protocol provided below. Include a photograph of your gel in the Word document that you started last week. Safety & hazardous materials GelStar is thought to be carcinogenic. Use caution and be sure to wear gloves when handling. UV light can permanently damage your eyes. Be sure to wear UV-protective goggles when performing excising bands from agarose gels. Materials Reagents Qiaex II Gel Extraction Kit (Qiagen 500preps/$316) Generuler 1Kb DNA ladder (Fermentas SM1163 $140) 6x TriTrack Loading Dye solution (Fermentas R1161 $32) 50x TAE Prepare 500mL of 50x TAE as follows: 121g Tris Base, 28.6ml glacial acetic acid, 50ml 0.5M EDTA ph 8.0, QC to 500ml. Autoclave and store at RT. 1x TAE QC 10ml 50x TAE to 500ml in dh 2 O. Autoclave and store at RT. GelStar Nucleic Acid Stain (Lonza x250µl $164.48) or GelGreen Nucleic Acid Stain (Phenix RGB x500µl $95.90) 1 Vogelstein, B., Gillespie, D., (1979) Preparative and analytical purification of DNA from agarose, Proc. Natl. Acad. Sci. USA 76: BIO-360 Genetics Lab 3 PCR amplification & gel extraction 1

2 Electronic heat block or water bath Microcentrifuge Accuprime Taq Polymerase (Invitrogen R $239) or AccuPrime GC-Rich Pyro Polymerase (Invitrogen R $313) Introduction to polymerase chain reaction (PCR) In 1983, Kary Mullis at Cetus Corporation developed a molecular biology technique that has since revolutionized genetic research and earned him the Nobel Prize in This technique, termed the polymerase chain reaction (PCR), was rapidly adopted as a significant multidisciplinary research tool. Before the invention of PCR, techniques for genetic analysis were labor intensive, time consuming, and required a high level of technical expertise. PCR has contributed to the development and popularization of gene maping, gene cloning, DNA sequencing, and gene detection technology. The objective of PCR is to produce a relatively large amount of a specific piece of DNA from a small amount of nonspecific DNA. Technically speaking, this means the controlled enzymatic amplification of a template DNA molecule containing a specific DNA sequence of interest. A researcher PCR to amplify trace amounts of DNA from a drop of blood or a single hair follicle to generate millions of copies of a desired DNA fragment. In theory, only one template strand is needed to generate millions of new DNA molecules. Prior to PCR, genetic and forensic analysis required copious amounts of DNA. PCR Makes Use of Two Basic Processes in Molecular Genetics: 1. Complementary DNA strand hybridization 2. DNA strand synthesis via DNA polymerase Figure 1. A recent photo of Kary Mullis (karymullis.com) Before a region of DNA can be amplified, one must identify and determine the sequence of a piece of DNA upstream and downstream of the region of interest. These areas are then used to make the oligonucleotide primers that will serve as starting points for DNA replication. Again, primers are needed because DNA polymerases require double-stranded DNA (as opposed to single stranded DNA) to ini- BIO-360 Genetics Lab 3 PCR amplification & gel extraction 2

3 tiate replication of DNA or synthesize new copies of template DNA. The two strands of the fragment of interest will be melted apart into single strands before synthesis begins. Therefore, primers are required to provide a double-stranded start point for the DNA polymerase. The DNA polymerase used in PCR, however, must be a thermally stable polymerase because the polymerase chain reaction cycles between temperatures of ~60 C and ~94 C. A thermostable DNA polymerase (Taq polymerase) from the thermophilic bacterium, Thermus aquaticus, is commonly used for this purpose. Alternatively, it has been recently demonstrated that the DNA polymerase (Pyro polymerase) from the archaebacterium Pyrolobus fumarius while equally thermostable, retains activity longer and demonstrates greater processivity (average number of nucleotides added per association). Following sample preparation, the template DNA, oligonucleotide primers, thermostable DNA polymerase, the four deoxynucleotides (A, T, G, C), and reaction buffer are mixed in a single microfuge tube. The tube is placed into the thermal cycler. Thermal cyclers contain an aluminum block that holds the samples and can be rapidly heated and cooled across extreme temperature differences. The first step of the PCR temperature cycling procedure involves heating the sample to 94 C. At this high temperature the template strands separate (denature). This is called the denaturation step. The thermal cycler then rapidly drops the temperature to C to allow the primers to anneal to the separated template strands. This is called the annealing step. There is the possibility that the two original template strands will reanneal to each other or compete with the primers for the primers complementary binding site. However, the oligonucleotide primers are added in excess such that the primers actually out compete the original DNA strand for the primers' complementary binding sites. Lastly, the thermal cycler heats the sample to 72 C for the DNA polymerase to extend the primers and make complete copies of each template DNA strand. Becaues the polymerase works most efficiently at this temperature it is called the extension step. Two new copies of each complementary strand are created. There are now two sets of template strands. These two sets of template strands can now be used for another temperature/thermal cycle and subsequent strand synthesis. At this stage, a complete temperature cycle (thermal cycle) has been completed (Figure 2). Temperature cycle = denaturation step + annealing step + extension step BIO-360 Genetics Lab 3 PCR amplification & gel extraction 3

4 Fig. 2. A complete cycle of PCR. Thermal cycling continues for 40 cycles. After each thermal cycle the number of template strands doubles, resulting in an exponential increase in the number of template DNA strands. After 40 cycles there will be 1.1 x more copies of the original number of template DNA molecules. The most unique feature of PCR is the generation of a precise length and sequence of DNA. On the first cycle the two different oligonucleotide primers anneal to the original genomic template DNA strands at opposite ends and on opposite strands. After the first complete temperature cycle, two new strands are generated that are shorter than the original template strands but still longer than the length of the DNA that the researcher wants to amplify. It isn t until the third thermal cycle that fragments of the precise length are generated (see Figure 3). BIO-360 Genetics Lab 3 PCR amplification & gel extraction 4

5 Fig. 3. Generation of precise length fragments. It is the template strands of the precise length that are amplified exponentially (X n, where X = the number of original template strands and n = the number of cycles). There is always one set of original long-template DNA molecules which is never fully duplicated. After each thermal cycle, two intermediate length strands are produced, but because they can only be generated from the original template strands, the intermediate strands are not exponentially amplified. It is the precise length strands generated from the intermediate strands that amplify exponentially at each cycle. Therefore, if 20 thermal cycles were conducted from one double stranded DNA molecule, there would be 1 set of original genomic template DNA strands, 20 sets of intermediate template strands, and 1,048,555 sets of precise length template strands. After 40 cycles there would be 1 set of original genomic template DNA strands, 40 sets of intermediate template strands, and 1.1 x sets of precise length template strands (see Figure 4). BIO-360 Genetics Lab 3 PCR amplification & gel extraction 5

6 Fig. 4. Schematic of PCR amplification of DNA fragments. Oligonucleotide primer dilution The oligonucleotide primers that you designed and were ordered from Integrated DNA Technologies, Inc. (idtdna.com) are lyophilized (freeze-dried) for stability at room temperatures. So we must first reconstitute the primers in DNase-free water or other suitable buffer. NOTE: When preparing primer stock solutions always use aerosol-barrier pipette tips to prevent the cross contamination through transfer of aerosolized sample. 1. Briefly spin the microfuge tube containing your oligonucleotide primer to ensure that it is all at the bottom of your tube. 2. Dilute your oligonucleotide primers in molecular biology grade water to create a 100µM stock solution of each primer. An easy way to calculate the amount of water needed here is to multiple the number (in nmol) of primer listed on your tube by 10. This is the volume of water in µl that you should add to the tube. Briefly vortex the solution. 3. Create a 10µM primer working solution by adding 10µL of the sense forward primer and 10µL of the antisense reverse primer to 80µL of molecular biology grade water. Briefly vortex the solution. Be sure to label the tube well. Store all of these tubes in a -20 o C freezer. BIO-360 Genetics Lab 3 PCR amplification & gel extraction 6

7 Polymerase chain reaction (PCR) NOTE: When setting up PCR reactions only use a fresh aerosol-barrier pipette tip to obtain the requsite enzyme and buffer from the stock solutions. Avoid contamination of shared stock solutions at all cost! PCR reactions commonly suffer from the amplification of nonspecific sequences. To reduce this risk, many commercial suppliers of PCR polymerase enzymes bind the enzyme with an antibody that is released only when the reaction is heated to 94 o C during the denaturation step of the first cycle. Such a hot-start polymerase inhibit the activity of the enzyme while the reaction tubes are being set up. Additionally, proprietary proteins have been developed which may further enhance the primer specificity during each cycle. Both the Accuprime Taq polymerase system and the Accuprine GC-Rich Pyro polymerase systems from Invitrogen incorporates these technologies. 1. Prepare the following 25µL PCR reactions in each of four thin-wall 0.2mL PCR tubes (a master mix may be used here). Standard Accuprime Taq Polymerase Reaction 21.0 µl Water (molecular biology grade) 2.5 µl 10x Accuprime polymerase buffer II (contains MgCl 2 & dntps) 0.5 µl Genomic DNA in TE (~0.10µg) 0.5 µl Primer mix working sol (10µM each) 0.5 µl Accuprime Taq polymerase GC-Rich Accuprime Pyro Polymerase Reaction 18.5 µl Water (molecular biology grade) 5 µl 5x Buffer A (for GC-rich templates) or 5x Buffer B 0.5 µl Genomic DNA in TE (~0.10µg) 0.5 µl Primer mix working sol (10µM each) 0.5 µl Accuprime Pyro polymerase 2. Run following PCR program as shown below (also see Figure 5): Temperature Time 95 o C 3 min ( Hotstart polymerase activation) 40 cycles 95 o C 30 sec (template denaturation) o C 30 sec (primer annealing) 72 o C 1 min/1kp amplicon length (extension) End cycles 72 o C 2-10min (final extension) BIO-360 Genetics Lab 3 PCR amplification & gel extraction 7

8 4 o C Infinity (inhibits amplicon degredation) Fig. 5. The programming screen of a Bio-Rad thermocycler. Note that the primer annealing step is set in gradient mode. This means that each column will be set at an independent annealing temperature. This setting is useful if the optimal annealing temperature has yet to be empirically determined. 3. Carefully label your PCR product and store it at -20 o C for characterization and cloning during lab 3. Agarose gel purification of PCR amplicons Electrophoretic separation of PCR reaction product allows one to characterize the reaction specificity and confirm that the amplicon length is correct. Subsequently, the amplicon may be excised from the gel and purified. 1. Prepare an agarose gel of a concentration appropriate for your amplicon (see Table 1 for selection of agarose gel concentration) by heating agarose in 50ml 1xTAE in the microwave until completely dissolved (~30sec to 1min). Use caution to not burn yourself or boil the solution over. % agarose Range of effective Bromophenol Orange G separation, bp blue Xylene cyanol 0.5 2,000-50,000 1,150 16, , , ,000 ~ , , , , , ,000 ~ , Table 1. Recommended agarose gel concentration for separation of linear DNA corresponding migration rates of marker dyes. 2. Add GelGreen or GelStar DNA stain to 0.5x concentration (2.5µl per 50ml gel). BIO-360 Genetics Lab 3 PCR amplification & gel extraction 8

9 3. Pour gel and place comb in one end. Allow to solidify. 4. Remove comb and submerge in 1x TAE buffer. Use a minimal amount of buffer to cover gel to maximize the rate of electrophoresis and conserve TAE (this requires ~275ml of 1x TAE buffer). 5. Load 5µl (0.5µg) of the GeneRuler DNA 1Kb DNA ladder (see FIgure 6) in the first lane of the gel. Figure 6. 1Kb DNA Ladder. Adopted from the 2007 Fermentas Catalog p Combine 5ul of your DNA solution with 1ul 6x TriTrack loading dye (see Figure 2) and load onto the second lane of the gel. 7. Check that the wells of the agarose gels are near the black ( ) electrode and the base of the gel is near the red (+) electrode. The negatively charged DNA and tracking dyes will migrate toward the red (+) electrode. Electrophorese the gel at ~100v (~7v/cm) until the bromophenol blue is near the end of the gel (~30-60min). 8. Image gel using the Kodak IS4000MM image station. Be sure to save a copy of this photo in your electronic laboratory notebook and annotate the photo appropriately. Introduction to DNA extraction from agarose In the presence of chaotropic salts (in this case, sodium iodide) DNA binds to glass particles. The chaotropic salts and agarose and other impurities are washed from the glass particles containing adsorbed DNA. The washing steps are followed by elution of the DNA in TE buffer or water. This method can be used to: BIO-360 Genetics Lab 3 PCR amplification & gel extraction 9

10 -purify DNA fragment from any type of agarose -concentrate DNA -remove proteins (eg following a restriction enzyme digest) -remove unincorporated nucleotides, primer, primer-dimers and enzymes from a PCR reaction -remove gel-star/gel-green or ethidium bromide from DNA This reaction is inhibited by TBE (Tris-borate-EDTA) buffer, be sure to use TAE buffer only for electrophoresis if you intend to gel purify. This method works with double stranded DNA greater than ~100bp (will not purify bind most primer-dimers). However, care should be taken to prevent shredding of DNA larger than 5kbp. DNA adsorption onto the silica gel is ph dependant. The Qiaex II kit contains a ph indicator. Optimal DNA binding only occurs when the indicator dye is yellow (ph<7.5). If the buffer appears purple, the high ph will inhibit DNA binding consult your instructor. Amplicon extraction protocol 1. Excise the gel slice containing the amplicon band with a clean scalpel (see Figure 7). Minimize the time of UV exposure as much as it is practical, as this will cause mutations. Minimize the size of the gel slice by trimming excess agarose. CAUTION: Use gloves to avoid exposure of your skin to mutagenic Gel-Star and wear UV-blocking goggles to avoid exposure of your eyes to damaging UV light! Figure 7. Excision of the correct DNA band from an agarose gel. 2. Determine an approximate volume of gel slice by weight (1g equals approximately 1ml) by weighing the gel slice in a 1.5mL plastic microfuge tube. Be sure to weigh tube before adding the gel slice, so that you can determine the mass of the gel by subtraction. BIO-360 Genetics Lab 3 PCR amplification & gel extraction 10

11 3. Add 3 volumes of Buffer QX1 DNA binding solution to 1 volume of gel. Incubate 5 minutes at 55 C to dissolve agarose. 4. Resuspend the Qiaex II silica gel suspension by vortexing for 30 sec. Add 10µL of the resuspended Qiaex II to the tube containing your gel slice. 5. Incubate at 50 o C for 10min to solubilize the agarose and bind the DNA to the silica. Mix by vortexing every 2min to kepp the Qiaex II in suspension. Be sure that the color of the mixture is yellow! If it is purple, you must imediately add 10µL 3M sodium acetate, ph 5.0 to adjust the ph. 6. Centrifuge the sample for 30sec and carefully digard the supernatent with a pipet. 7. Wash the pellet with 500µL of wash buffer QX1. Resuspend by vortexing, pellet with a brief centrifugation, and remove the supernatent with a pipet. This step removes residual agarose. 8. Wash the pellet with twice with 500µL of wash buffer PE. Resuspend by vortexing, pellet with a brief centrifugation, and remove the supernatent with a pipet. These steps remove residual salts. 9. Air-dry the pellet for min or until the pellet appears white. Do not overdry, as this will decrease elution efficiency. 10. Elute DNA into TE buffer (ph 8.0). Resuspend the pellet in a 30µL of sterile TE buffer and incubate the tube at 55 C for 5 minutes. Spin the tube and remove the supernatant into a new microfuge tube, taking care to avoid the pellet. 8. Store the DNA in a well-labeled tube at -20 o C. BIO-360 Genetics Lab 3 PCR amplification & gel extraction 11

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