Isolation of genomic DNA from buccal swabs - a brief protocol. Assessment of DNA concentration and purity

Transcription

1 Molecular biology 1 DNA Isolation Isolation of genomic DNA from buccal swabs - a brief protocol MACHEREY-NAGEL isolation kit Protocol: 1. Gently rub and rotate swab along the inside of the cheek (both left and right side), ensuring that the entire swab tip has made contact with the cheek, approx. 2-3 min. During removing the swab from mouth, be careful not to touch swab tip against teeth, lips, or other surface. 2. Put the swab tip into 1.5mL eppendorf tube and break the wooden stem so that the part with cotton stays in the eppendorf tube. 3. Add 100 L PBS, 15 L proteinase K, 100 L Buffer B3. Vortex vigorously for 60s. 4. Put the tube into thermoblock and incubate at 56 C for 10 min. Vortex for 30s. 5. Put the tube into thermoblock and incubate at 70 C for 5 min. Vortex for 30s. 6. Add 100 L 96% ethanol and vortex for 10s. Spin down shortly. 7. Load the lysate (without cotton part of the forensic swab) onto the NucleoSpin Column. Centrifuge 1 min at 12,000 RPM. Discard Colection Tube with flow-through. 8. Place the NucleoSpin Column into a new Collection Tube and add 400 L Buffer BW. Centrifuge 1 min at 12,000 RPM. Discard Colection Tube with flow-through. 9. Place the NucleoSpin Column into a new Collection Tube and add 400 L Buffer B5 Centrifuge 1 min at 12,000 RPM. Discard the flow-through and reuse Collection Tube. 10. Place the NucleoSpin Column back into the Collection Tube and centrifuge 3 min at 12,000 RPM. 11. Place the NucleoSpin Column in a new 1.5mL eppendorf tube and add carefully 50 L preheated Buffer BE (70 C). Dispense the buffer directly onto the silica membrane! Incubate at room temperature for 1 min. 12. Centrifuge 1 min at 12,000 RPM. Assessment of DNA concentration and purity There are several methods to determine the concentration of nucleic acids as well as their purity - spectrophotometry (absorbance measurement), agarose gel electrophoresis, fluorometry using fluorescent DNA-binding dyes. In our practical classes, we will use spectrophotometric estimation. Nucleic acids absorb ultraviolet light with an absorption maximum at 260 nm, proteins at 280 nm. Low molecular weight substances (e.g. phenol, chloroform, EDTA) have its absorption maximum at 230 nm. The nucleic acid concentration is calculated from the absorbance measured at 260 nm. A 260 = 1 corresponds to: double stranded DNA (dsdna) at concentration 50 µg/ml single stranded DNA (ssdna) at concentration 37 µg/ml RNA at concentration 40 µg/ml The ratios A 260 /A 280 and A 260 /A 230 are used to assess the purity of nucleic acids. The ratio A 260 /A 280 should be for pure DNA around 1.8, for pure RNA around 2. Lower A 260 /A 280 values may indicate protein contamination. The ratio A 260 /A 230 should be for pure DNA higher than 2.0. Lower A 260 /A 230 values indicate contamination with salts or solvents, such as phenol. Residual chemical contamination from 1

2 Molecular biology 1 DNA Isolation nucleic acids extraction procedures may result an overestimation of the nucleic acid concentration and/or negatively influence downstream analysis. If the required purity is not met, reprecipitation of the sample is required, resulting in a significant reduction in the impurity content. Protocol: DeNovix DS-11 microvolume Spectrometer - apply 1 μl of sample. 1. Select the program: "dsdna" on the spectrophotometer. 2. Set the Spectrophotometer against the blank solution ("blank") - in our case BE Buffer: Ensure both top and bottom sample surfaces are clean. Pipette 1 μl of BE Buffer onto the lower sample surface, lower the top arm and press the BLANK button. Then open the lid, remove the solution from both sample surfaces using a clean, dry lab wipe. 3. Measure the absorption spectrum of the sample within the range 220 nm nm: Pipette 1 μl of sample onto the lower sample surface, lower the top arm and press the MEASURE button. Then open the lid, remove the solution from both sample surfaces using a clean, dry lab wipe. 4. The device displays the measured spectrum, DNA concentration, and the A 260 /A 280 and A 260 /A 230 ratios that give us information about the purity of the solution. 5. Print an overview of the measured values. A) Pipetting sample solution onto pedestal. B) Measurement. 2

3 Molecular biology 2 Polymerase chain reaction (PCR) Polymerase chain reaction - a brief protocol In this laboratory exercise, we will amplify a region of the Factor V gene (the region which contains SNP of our interest) using Polymerase Chain Reaction (PCR). Wear gloves (powder-free)! Fill a polystyrene box with ice from the icemaker. Keep the reagents on ice always when possible. Master Mix Preparation The total volume for a single PCR reaction is very small (20 µl). It would be uncomfortable to pipette separately all the components needed for each PCR reaction. The strategy is to prepare so called Master Mix which contains all the common components for a set of reactions. It improves consistency among the reactions and reduces pipetting error. Take a new 1.5mL eppendorf tube and prepare the Master Mix for 10 PCR reactions, each of the total volume 20 L (19 L Master Mix + 1 L DNA sample). water PCR buffer (10x) 20 Mg 2+ 5 dntp 4 forward primer (F) 2 reverse primer (R) 2 Taq polymerase 2 enzyme freezer! L DNA sample 10 DNA sample will be added later into individual PCR test tubes Vortex the eppendorf tube with Master Mix for 10s. Spin down shortly. Keep on ice. Setting up PCR reactions Take out your DNA sample from the freezer and let it melt at the room temperature. Prepare 10 PCR test tubes. Dispense 19 L of the Master Mix into each tube. Add 1 L of the DNA sample. sample sample sample sample sample sample sample sample posit. neg control control Put the PCR tubes into the block of thermocycler and start up the predefined program. PCR Parameters: 1) Initial denaturation: 95 C - 5 min 2) 35 cycles of: 95 C - 15 s 60 C - 15 s 72 C - 30 s 3) Linked to: 72 C - 7 min 4) Linked to: 4 C - indefinitel 3

4 Molecular biology 3 Restriction enzyme analysis + gel electrophoresis + interpretation of the results In this laboratory exercise, you will use the restriction endonuclease MnlI for analysis of the DNA fragment amplified by PCR method last time. Wear gloves (powder-free)! Restriction enzyme analysis You will receive 3 PCR test tubes from the set of ten PCR reactions processed last time. There is 20 L of PCR reaction mixture (hopefully with the PCR product) in each tube. your DNA sample test tube marked with your initials positive control (factor V Leiden, heterozygote) test tube marked "+K" negative control test tube marked " K" To perform restriction enzyme analysis, take two new PCR test tubes and tranfer exactly 10 µl of the PCR product from the test tubes with your DNA sample and positive control into the new test tubes. Mark the new test tubes with symbol "R", i.e. "your initials R", "+KR". Restriction analysis 10 µl 10 µl PCR products sample posit. neg. control control The total volume of restriction analysis reaction will be 20 L. Now there is 10 L of the PCR product. 1. Add the following components in the order indicated: PCR product (DNA) 10 µl water 7 µl 10x FastDigest Green Buffer 2 µl FastDigest enzyme MnlI 1 µl L 2. Mix gently and spin down. 3. Incubate at 37 C in a heat block of the thermocycler for 10 min. 4

5 Molecular biology 3 Restriction enzyme analysis + gel electrophoresis + interpretation of the results Gel electrophoresis Add 2 L of loading dye into the three test tubes with PCR products. Load 10 L of the reaction mixtures on a gel as demonstrated in the figure. There are 13 well in the gel we use. Each group of students will use one half of the gel (6 wells as indicated). Interpretation of the results length of PCR product: 288 bp restriction analysis: homozygote wild type: 158 bp, 93 bp, 37 bp factor V Leiden heterozygote: 158 bp, 130 bp, 93 bp, 37 bp factor V Leiden homozygote: 158 bp, 130 bp 5