Presentation on theme: "Uji kualitatif dan kuantitatif DNA dan RNA Fatchiyah, PhD JBUB,"— Presentation transcript:
Uji kualitatif dan kuantitatif DNA dan RNA Fatchiyah, PhD JBUB,
Uji Kuantitatif Uji kuantitatif DNA dengan spektrofotometri UV-Vis, DNA murni dapat menyerap cahaya ultraviolet karena keberadaan basa-basa purin dan pirimidin. Pita ganda DNA dapat menyerap cahaya UV pada 260 nm, sedang kontaminan protein atau phenol akan menyerap cahaya pada 280 nm. Sehingga kemurnian DNA dapat dukur dengan menghitung nilai absorbansi 260 nm dibagi dengan nilai absorbansi 280 (Å260/Å280), dan nilai kemurnian DNA berkisar antara NanoDrop
Mengukur Konsentrasi DNA/RNA Serta untuk mengukur konsentrasi DNA digunakan rumus sebagai berikut: [DNA] = Å260 x 50 x faktor pengenceran Å 260 = Nilai absorbansi pada 260 nm 50 = larutan dengan nilai absorbansi 1.0 sebanding dengan 50 ug untai ganda DNA per ml (dsDNA) [RNA] = Å260 x 40 x faktor pengenceran 40 = 40ug/ml untai tunggal RNA (ssRNA)
Uji Kualitatif Metoda standar yang digunakan untuk memisahkan, mengidentifikasi dan memurnikan fragmen DNA adalah elektroforesis gel agorose. Teknik ini sederhana, cepat terbentuk, dan mampu memisahkan campuran potongan DNA sesuai dengan ukurannya secara akurat, dibanding dengan densitas gradient sentrifugasi. Selanjutnya, lokasi DNA dalam gel tersebut dapat diidentifikasi secara langsung dengan menggunakan pewarna berfluorescen. 4/12/2015fatchiyah, JB-UB4
Electrophoresis for nucleic acid Agarose Gel Electrophoresis Purification for Specific Fragment of DNA -DNA Electro-elution -Electrophoresis onto DEAE-cellulose membranes Polyacrylamide Gels Pulse-field Gel Electrophoresis (PFGE) 4/12/2015fatchiyah, JB-UB5
Preparing and Running Standard Agarose DNA Gels The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively simple and include: An electrophoresis chamber and power supply Gel casting trays, which are available in a variety of sizes and composed of UV- transparent plastic. The open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis. Sample combs, around which molten agarose is poured to form sample wells in the gel. 4/12/2015fatchiyah, JB-UB6
Preparing and Running Standard Agarose DNA Gels Electrophoresis buffer, usually Tris- acetate-EDTA (TAE) or Tris-borate-EDTA (TBE). Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded.
Preparation of Gel 4/12/2015fatchiyah, JB-UB8 Ethidium bromide, a fluorescent dye used for staining nucleic acids. NOTE: Ethidium bromide is a known mutagen and should be handled as a hazardous chemical - wear gloves while handling. Transilluminator (an ultraviolet lightbox), which is used to visualize ethidium bromide-stained DNA in gels. NOTE: always wear protective eyewear when observing DNA on a transilluminator to prevent damage to the eyes from UV light.
Tabel 1. konsentrasi gel agarose dan ukuran molekul DNA NoKonsentrasi Gel Agarose (%) Effisiensi range Pemisahan pada DNA linier (kb)
DNA and RNA molecules are negatively charged, thus move in the gel matrix toward the positive pole (+) Linear DNA molecules are separated according to size The mobility of circular DNA molecules is affected by their topological structures. The mobility of the same molecular weight DNA molecule with different shapes is: supercoiled> linear> nicked or relaxed 4/12/2015fatchiyah, JB-UB10 Chemistry of nucleic acids
Migration of DNA Fragments in Agarose Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight. In other words, if you plot the distance from the well that DNA fragments have migrated against the log10 of either their molecular weights or number of base pairs, a roughly straight line will appear. 4/12/2015fatchiyah, JB-UB11
Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass. Typically, uncut plasmids will appear to migrate more rapidly than the same plasmid when linearized. Additionally, most preparations of uncut plasmid contain at least two topologically-different forms of DNA, corresponding to supercoiled forms and nicked circles. The image to the right shows an ethidium- stained gel with uncut plasmid in the left lane and the same plasmid linearized at a single site in the right lane.
DNA Migration 4/12/2015fatchiyah, JB-UB14 largemoderatesmall Picture of DNA separation by gel electrophoresis M S
Factors of DNA Migration Several additional factors have important effects on the mobility of DNA fragments in agarose gels, and can be used to your advantage in optimizing separation of DNA fragments. Chief among these factors are: Agarose Concentration Voltage Electrophoresis buffer Effects of Ethidium Bromide 4/12/2015fatchiyah, JB-UB15 Fig. Agarose Concentration
Fig. 13-2, p.331
Fig. 13-1, p.331
Purification for Specific Fragment of DNA In addition to its importance as an analytical tool, gel electrophoresis is widely used for isolating and then purifying specific fragments of DNA, usually in preparation for subcloning Several techniques can be used to purify DNA from agarose gels, and choosing between them is, to some extent, a matter of personal preference. They all start out by excising the desired "band" from an ethidium-stained gel viewed with a UV transilluminator. Because UV light can fragment DNA, it is best to work expeditiously and keep exposure time to a minimum. 4/12/2015fatchiyah, JB-UB19
DNA Electroelution Cut out the desired piece of agarose using a razor blade or scalpel blade, and try to get as little extra agarose as possible. The block of agarose containing DNA is then subjected to any of the following. The block of agarose is placed in a piece of dialysis tubing with a small amount of fresh electrophoresis buffer, the ends sealed with clamps, and the bag placed into an electrophoresis chamber. Application of current will cause the DNA to migrate out of the agarose, but it will be trapped within the bag. 4/12/2015fatchiyah, JB-UB20
DNA Electroelution.. 4/12/2015fatchiyah, JB-UB21 Progress can be monitored using a transilluminator, as shown below. When the DNA is out of the agarose, the flow of current is reversed for a few seconds to knock the DNA off of the side of the tubing. The buffer containing the DNA is then collected and the DNA precipitated with ethanol. Electroelution is more time consuming than some of the other techniques, but works well and is probably the best technique for recovery of large (> 5 kb) fragments of DNA.
22 To separate DNA of different size ranges Narrow size range of DNA: use polyacrylamide Wide size range of DNA: use agarose gel Very large DNA(>30-50kb): use pulsed-field gel electrophoresis Electrophoresis
Electrophoresis onto DEAE- cellulose membranes At low concentrations of salt, DNA binds avidly to DEAE- cellulose membranes. Fragments of DNA are electrophoresed in a standard agarose gel until they resolve adequately. One then makes a slit in the gel slightly ahead of the fragment(s) of interest and resumes electrophoresis until all of that fragment has migrated and stuck onto the membrane. 4/12/2015fatchiyah, JB-UB23
Electrophoresis onto DEAE- cellulose membranes The membrane is then removed, washed free of agarose in low salt buffer (150 mM NaCl, 50 mM Tris, 10 mM EDTA), then incubated for about 30 minutes at 65 C in high salt buffer (1 M NaCl, 50 mM Tris, 10 mM EDTA) to elute the DNA. Progress in binding DNA to the membrane and eluting it can be monitored with UV light to detect the ethidium bromide bound to DNA. After elution, DNA is precipitated with ethanol. This procedure is simple and provides very clean DNA. However, fragments larger than about 5 kb do not elute well from the membrane.
Strand-separating Gels For some purposes, eg. sequencing by Maxam-Gilbert procedure. It is necessary to obtain separated stands of fragment of DNA. Often this can be achieved by electrophoresis of denatured DNA through neutral agarose. The strands of DNA fragment less than 1kb in length are separated on polyacrilamide gel. Polyacrylamide gel necessary to obtain separated the each nucleotide of DNA sequence 4/12/2015fatchiyah, JB-UB25
Polyacrylamide Gels A commonly-used means of recovering DNA from polyacrylamide gels is by the so-called "crush and soak" method. The slice of polyacrylamide containing DNA is crushed in a microcentrifuge using a plastic pipet tip, and incubated with constant shaking in elution buffer (high salt) at 37ºC for several hours. The polyacrylamide pieces are then eliminated by centrifugation or by passing the mixture through a plug of siliconized glass wool. Finally, DNA is recovered by ethanol precipitation. DNA can also be recovered from polyacrylamide by use of certain types of silica gel particles, as described above for recovery from agarose. However, small (< 100 bp) fragments of DNA are very difficult to elute from standard glass particles. 4/12/2015fatchiyah, JB-UB27
Pulse-field gel Electrophoresis (PFGE) Ideally, the DNA should separate in straight lanes to simplify lane-to-lane comparisons. The original pulsed-field systems used inhomogeneous electric fields that did not produce straight lanes, making interpretation of gels difficult (Schwartz and Cantor, 1984). Again, the simplest approach to straight lanes is FIGE, which uses parallel electrodes to assure a homogeneous electric field. Although extremely useful for separating relatively small DNA, 4- 1,000 kb (fig. 2), FIGE's reorientation angle of 180ø results in a separation range most useful under 2,000 kb. Furthermore, like other PFGE techniques, FIGE has mobility inversions in which larger DNA can move ahead of smaller DNA during electrophoresis. 4/12/2015fatchiyah, JB-UB28
29 pulsed-field gel electrophoresis Switching between two orientations: the larger the DNA is, the longer it takes to reorient Electrophoresis
PFGE System: Electrode Configuration 4/12/2015fatchiyah, JB-UB30 Figure 1:Electrode configuration of commonly used pulsed field gel electrophpresis units.
PFGE Result Figure 2. Increased separation of the kb range with field inversion gel electrophoresis (FIGE). Run conditions: 230 V, 7.9 V/cm, 16 hrs., 50 msec. pulse, forward:reverse pulse ratio = 2.5:1, 1% GTG agarose, 0.5X TBE, 10 C.a) 1 kb ladder, kb; b) Lambda/Hind III, kb; and c) High molecular weight markers, kb. 4/12/2015fatchiyah, JB-UB31