Short Tandam Repeat (D1S58) Detection Kit (for Academic Instructions) Product # 54600

Transcription

1 3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: (905) Fax: (905) Short Tandam Repeat (D1S58) Detection Kit (for Academic Instructions) Product # Product Insert DNA (deoxyribonucleic Acid) is the genetic material that determines a person's biological characteristics. The complete human genome is stored as DNA sequences within the chromosome pairs found in the cells in a human body. A person s DNA is acquired from both parents; one copy of each of chromosome comes from the mother and one copy come from the father. As DNA is inheritable, it becomes an important material used in human identification. This is achieved by comparing one's DNA profile with that of a parent, child, sibling or other relatives. A number of DNA identification systems have been developed over the years. Many of the developed tests utilize genetic markers known as Short Tandam Repeats (STRs). STRs are repeating sequences (from 2 to 20 bp) of DNA. Loci with these STRs could be highly polymorphic, resulting in a number of alleles that contain variable numbers of the STRs. In fact, most common databases of DNA fingerprinting (including that of the FBI) utilize the Combined DNA Index System (CODIS) markers and analyze 15 standard STR genetic markers (plus a 16th marker for sex determination). The use of STR genetic markers in human identification is very powerful and could be used to provide accurate relationship determination, including for paternity tests. Principle of the Test and Product Description Norgen s Short Tandam Repeat (D1S58) Detection Kit uses PCR to determine the genotype of a human Short Tandam Repeat (STR) polymorphism at the D1S58 locus. The D1S58 locus is present in the Human Chromosome 1. It contains a 16 bp repeating unit with a core sequence of G-N-P-G-A-C- C-A-C-(A or C)-G-G-N-A-A-G (N is any nucleotide base and P is any purine base, A or G). The locus is highly polymorphic with the reported number of repeating units ranging from 13 to 44 (with a molecular weight of 354 bp to 850 bp including the flanking sequences). This kit protocol is a multi-part procedure. First, genomic DNA is isolated from the individual(s), and this can be achieved by using Norgen's Saliva DNA Collection, Preservation and Isolation Kit (Cat# RU35700). The genomic DNA is then subjected to the PCR reactions to amplify the D1S58 locus. The products from the PCR reactions are then resolved and visualized using standard DNA agarose gel electrophoresis. A 100 bp DNA Ladder as well as a D1S58 Allelic Ladder is also provided. The migration pattern (distance migrated by each band on the gel) of both the ladders will be used to construct a standard curve. The migration pattern of the PCR product(s) for each individual is also measured and recorded. The number of repeating units will then be determined using the standard curve constructed. Kit Components: Component Contents 2x PCR Master Mix with Dual Dye 0.5 ml 10x D1S58 STR Detection Primer Mix 30 μl Nuclease-Free Water 1.25 ml Positive Control DNA a 10 μl D1S58 Allelic Ladder 20 μl PCR Sizer 100 bp DNA Ladder 100 μl 50x TAE Buffer 30 ml Agarose 5 g Product Insert 1 a The positive control is purified DNA fragments.

2 Recommended Product for DNA Isolation (not included in the kit): Saliva DNA Collection, Preservation and Isolation Kit (Norgen Biotek Cat# RU35700) Customer-Supplied Reagents and Equipment Disposable powder-free gloves Benchtop microcentrifuge Micropipettors Sterile pipette tips with filters PCR Strips and Caps Thermocycler / PCR Machine Agarose Gel Electrophoresis Apparatus and Power Supply (Optional) 10 mg/ml Ethidium Bromide (Cat# 28033) Storage Conditions and Product Stability All buffers should be kept tightly sealed and stored at room temperature (15-25 C). Buffers can be stored for up to 1 year without showing any reduction in performance. The 2x PCR Master Mix with Dual Dye, 10x D1S58 STR Detection Primer Mix, Positive Control DNA, and D1S58 Allelic Ladder should be kept tightly sealed and stored at -20 C. These can be stored for up to 1 year without showing any reduction in performance. Repeated thawing and freezing (> 2 x) of these reagents should be avoided, as this may reduce the sensitivity. If the reagents are to be used only intermittently, they should be frozen in aliquots. General Precautions The user should exercise the following precautions when using the kit: Use sterile pipette tips with filters. Store and extract positive material (specimens, controls and amplicons) separately from all other reagents and add it to the reaction mix in a spatially separated facility. Thaw all components thoroughly at room temperature before starting an assay. When thawed, mix the components and centrifuge briefly. Work quickly on ice. Quality Control In accordance with Norgen s ISO 9001 and ISO certified Quality Management System, each lot of Norgen s Short Tandam Repeat (D1S58) Detection Kit, including 2x PCR Master Mix with Dual Dye, 10x D1S58 STR Detection Primer Mix, Positive Control DNA, and D1S58 Allelic Ladder are tested against predetermined specifications to ensure consistent product quality. Product Warranty and Satisfaction Guarantee NORGEN BIOTEK CORPORATION guarantees the performance of all products in the manner described in our product manual. The customer must determine the suitability of the product for its particular use. Safety Information Wear gloves when performing the protocol. Refer to the MSDS sheets for safety procedures regarding reagents used.

3 Protocol Summary of Method The protocol is a multi-part procedure including: 1. Collection and purification of saliva DNA from the individual. 2. PCR Amplification of D1S58 locus from purified saliva DNA. 3. Resolution of PCR fragments on an agarose gel, together with molecular weight and allelic ladder. 4. Generation of Standard Curve by measuring the migration pattern of molecular weight and allelic ladder. 5. Determination of the number of STRs present in each individual using the Standard Curve. Section 1. Collection and Purification of Genomic DNA (Saliva) from Individual of Interest Notes: The recommended input source for DNA is Saliva. Additional guidelines for sample preparation are provided below. Other non-invasive inputs (such as buccal swab) could be used. However, the quality and the amount of sequence could vary, depending on the purity and the integrity of the genomic DNA isolated from these samples. Recommended Producst: Norgen s Saliva DNA Collection, Preservation and Isolation Kit (Cat# RU35700) - Contains Saliva Collection and Preservation Devices, as well as reagents for alcohol-precipitation-based DNA purification. Norgen s Saliva DNA Isolation Kit (Cat# RU45400) - Provides reagents for column-based DNA purification from saliva samples collected using Norgen s Saliva DNA Collection, Preservation and Isolation Kit (Cat# RU35700). Recommended Procedure: 1. Perform saliva collection and DNA isolation using Saliva DNA Collection, Preservation and Isolation Kit (Cat# RU35700). Follow the procedure for extraction of DNA from 0.5 ml of preserved saliva. 2. The average expected yield of DNA from 0.5 ml preserved saliva is ~ 20 μg. The average expected OD 260/280 ratio is ~ This could be verified using a standard spectrophotometer. 3. Based on the DNA concentration of the sample, dilute the DNA to approximately 10 ng/μl using nuclease-free water or TE. Proceed to Section 2. Section 2. PCR Amplification of the D1S58 Locus Notes: Before use, suitable amounts of all PCR components should be completely thawed at room temperature, vortexed and centrifuged briefly. The amount of 2x PCR Master Mix with Dual Dye, 10x D1S58 STR Detection Primer Mix, and Positive Control DNA provided is enough for tests of up to 8 individuals. A Positive Control DNA is included and could be applied directly to the procedure (see below). It is highly recommended that sterile pipette tips with filters be used to avoid any crosscontaminations. 1. Thaw the 2x PCR Master Mix with Dual Dye, 10x D1S58 STR Detection Primer Mix, and Positive Control DNA completely. Leave the thawed mixes on ice if the reaction is not set up immediate. 2. Set up 20 μl Sample PCR Reactions, as described in Table 1 below.

4 Table 1. Sample PCR Reaction Set up for D1S58 Components Nuclease-free Water 2X PCR Master Mix 10x D1S58 STR Detection Primer Mix Volume Reserved for DNA (Concentration at ~ 10 ng/ μl) Total Per 20 μl reaction 7 μl 10 μl 2 μl 1 μl 20 ul 3. Add each component (except the DNA template) of the PCR reaction, in the order shown in Table 1 into the PCR tube. Leave the tube, capped, on ice. 4. Add 1 μl of DNA to the PCR reaction mix. 5. Cap the PCR tube. Mix briefly. Label the PCR tube such that each test individual could be easily identified. 6. Repeat the set up for all test individuals. Leave the tubes, capped, on ice. 7. For each PCR run, Positive Control (PosC) and Negative Control (NegC) should be set up. 8. Set up Positive Control, as described in Table 2. Table 2. Positive Control PCR Reaction Set up Components Nuclease-free Water 2X PCR Master Mix 10x D1S58 STR Detection Primer Mix Positive Control DNA Total Per 20 μl reaction 7 μl 10 μl 2 μl 1 μl 20 ul 9. Add each component (except the PosC) of the PCR reaction, in the order shown in Table 2 into the PCR tube. Leave the tube, capped, on ice. 10. Add 1 μl of PosC to the PCR reaction mix. 11. Cap the PCR tube. Mix briefly. Label the PCR tube such that the Positive Control could be easily identified. Leave the tube, capped, on ice. 12. Set up Negative Controls, as described in Table 3. Table 3. Negative Control PCR Reaction Set up Components Nuclease-free Water 2X PCR Master Mix 10x D1S58 STR Detection Primer Mix Total Per 20 μl reaction 8 μl 10 μl 2 μl 20 μl

5 13. Add each component of the PCR reaction, in the order shown in Table 3 into the PCR tube. 14. Cap the PCR tube. Mix briefly. Label the PCR tube such that the Negative Control could be easily identified. 15. Load all samples into a thermocycler. Set up the PCR Cycling Time as below: 94 C for 2 minutes 1 Cycle 94 C for 10 seconds 65 C for 30 seconds 40 Cycles 72 C for 2 minutes 72 C for 10 minutes 1 Cycle Hold for 4 C 16. Proceed to Section 3 to resolve the PCR products on an agarose gel for visualization. Otherwise, store the reactions at -20 o C. Section 3. Resolution of the D1S58 PCR Products by Agarose Gel Electrophoresis Notes: Resolved DNA gels could be visualized by staining with Ethidium Bromide (EtBr). Caution: EtBr is a known mutagen. DNA gels stained with EtBr are to be visualized under ultraviolet (UV) light. Wear a lab coat, eye protection and gloves when working with this chemical. Alternatively, the DNA gel can be stained with a non-mutagenic stain and visualized according to its excitation/emission spectrum. A. Pouring a Standard 3 % (W/V) Agarose Gel: 1. Prepare 1 L of 1x TAE by diluting 20 ml of 50x TAE Buffer with 980 ml of deionized water. 2. Pour 100 ml of the 1x TAE into a microwavable flask. The remaining 900 ml 1x TAE could be set aside and used as running buffer for electrophoresis. 3. Measure 3 g of Agarose. 4. Pour the agarose powder into the microwavable flask containing the 100mL of 1xTAE. 5. Microwave for 1 to 3 minutes until the agarose is completely dissolved. Use a hot plate for boiling if a microwave is not available. Note: Caution HOT! Be careful when stirring, as eruptive boiling can occur. The most appropriate way to melt the agarose is to microwave for seconds, stop and swirl, and then continue towards a boil. Watch the flask closely as it has a tendency to boil over. 6. Allow the melted agarose solution to cool down for around 10 minutes. 7. (Optional) For Ethidium Bromide (EtBr) visualization, add 1 μl of 10 mg/ml Ethidium Bromide (EtBr) to the melted agarose solution and mix by swirling gently. Note: Caution: EtBr is a known mutagen. Wear a lab coat, eye protection and gloves when working with this chemical. Note: An alternative non-mutagenic stain could be used. Mix the stain with the agarose solution according to manufacturer's recommendation. 8. Pour the agarose into a gel tray with the well comb in place. Note: Ensure that there are enough wells for the PCR products as well as an additional 4 lanes for Positive Control, Negative Control, D1S58 Allelic Ladder and molecular weight ladder (1 well each) 9. Let the newly poured gel sit at room temperature for minutes, until it has completely solidified.

6 B. Loading Samples and Running the DNA Agarose Gel: 1. Once solidified, place the agarose gel into the appropriate gel box. Orient the gel in such a way that the loading well is closest to the negative electrode. Note: DNA is negatively charged and will run towards the positive electrode. In most gel boxes, the negative electrode is black while the positive electrode is red. 2. Fill the gel box with the remaining 1x TAE prepared in Step A2 above. Pour enough buffer to cover about 5 mm above the top of the gel. Note: Depending on the size of the gel box, an additional amount of 1x TAE may have to be prepared 3. Carefully load 10 μl of the PCR Sizer 100 bp Ladder, provided) into the first lane of the gel. Note: The PCR Sizer 100 bp Ladder is already pre-mixed with a blue loading dye. 4. Carefully load 10 μl of the D1S58 Allelic Ladder into the second lane of the gel, next to the molecular weight ladder. Note: The Allelic Ladder is already pre-mixed with a blue loading dye. 5. Carefully load 15 μl of the sample PCR product of the test individuals into the wells immediately next to the Allelic ladder. Note: The PCR reaction is already pre-mixed with an orange loading dye. 6. Load 15 μl of the PCR product of the Positive Control. 7. Load 15 μl of the Negative Control PCR. 8. Connect the gel box with the power supply appropriately. Connect the negative charge outlet of the power supply to the negative (black) electrode. Connect the positive charge outlet to the positive (red) electrode. 9. Set the power supply Voltage at constant at 150 V. Note: The current (Amp) will be variable. It could be set at the maximum amount available. 10. Start to run the gel at 150V for about 45 minutes Note: The loading dye of the PCR mix will be separated into two bands - the bigger band will have a dark red color (Cresol-Red, ~ 1 kbp) while the smaller band will have a light orange/yellow color (Orange G, ~ bp). The actual PCR products of D1S58 migrate between these two bands. Hence, the two colored bands could be used to track the movement of the PCR product. For the most optimal condition, allow the Orange G dye (light orange/yellow band) to migrate ~ 75-80% of the way down the gel. 11. Turn OFF power, disconnect the electrodes from the power supply, and then carefully remove the gel from the gel box. Note: It is desirable to place the gel in a suitable transport container or tray. 12. Perform the appropriate staining for DNA visualization. For EtBr Staining, use any device that has UV light to visualize your DNA fragments. Capture a picture of the DNA gel for record. Ensure that the loading wells are captured in the picture for subsequent migration distance measurement. Note: When using UV light, protect your eyes and skin by wearing safety goggles or a face shield, gloves and a lab coat.

7 C. Analyzing the Gel 1. Print out a copy of the picture of the DNA gel. 2. Label each lane appropriately. Clearly indicate the DNA molecular weight ladder, D1S58 Allelic Ladder, PCR products of each test individual, Positive Control and Negative Control. The PCR products have a molecular weight range of 354 bp to 850 bp. 3. An example of a test gel is shown below: MW AL NTC AL MW Figure 1. Rapid Allele Determination (Short Tandem Repeat numbers) of Human D1S58 Locus by End-Point PCR Norgen s Short Tandam Repeat (D1S58) Detection Kit (for Academic Instructions) allows for the rapid amplification of PCR products that could be used to distinguish the different alleles (different STR number) of the human D1S58 locus. For analysis, 20 μl PCR reactions (samples 1 to 5) and the No Template Control (NTC) were loaded on a 1X TAE 3% Agarose DNA gel with 10 μl of Norgen's PCR Sizer 100 bp DNA Ladder (150V for 30 minutes). A D1S58 Allelic Ladder with 8 DNA bands of known STR repeat number is also provided. The expected molecular weight range of the PCR product is from 354 bp to 850 bp, corresponding to 13 to 44 units. The PCR Sizer 100 bp Ladder (MW) as well as the D1S58 Allelic Ladder (AL) can be used to construct a standard curve to determine the number of STR units present in each test individual s PCR product(s). 4. Using a ruler, measure the distance between the bottom of the loading well and the bottom of each DNA band of the PCR Sizer 100 bp Ladder (MW). Record the migration distance results in the table provided below. MW, bp Distance Migrated from Loading Well, mm

8 5. Using a ruler, measure the distance between the bottom of the loading well and the bottom of each DNA band of the Allelic Ladder. Record the migration distance results in the table provided below. Allelic Ladder Band MW, bp Number of STR Unit Present Distance Migrated from Loading Well, mm 6. Using a ruler, measure the distance between the bottom of the loading well and the bottom of each DNA band of each test individual's PCR reaction. Each individual may contain one or two PCR products, depending whether he or she is homozygous or heterozygous for the D1S58 locus. Record the migration distance for both bands in the table provided below. Individual ID Larger Band's Distance Migrated from Loading Well, mm Smaller Band's Distance Migrated from Loading Well, mm

9 Distance Migrated, mm 7. Using the graph provided below, plot a Standard Curve by plotting the molecular weight (MW) of each band of the PCR Sizer 100 bp Ladder (MW) against the Distance Migrated, as recorded in Step C4. Determine the molecular weight of the PCR product(s) of each individual by applying the Distance Migrated by each band (recorded in Step 6) to the Standard Curve plotted. Record in the table below. Based on the Allelic Ladder information in Step 5, determine the number of STR units carried by each PCR band Molecular Weight, bp Individual ID 1. Larger Band's MW based on Standard Curve Larger Band's Number of STR Units Smaller Band's MW based on Standard Curve Smaller Band's Number of STR Units

10 Distance Migrated, mm 8. Using the graph provided below, plot a Standard Curve by plotting the number of STR units of each band of the Allelic Ladder against the Distance Migrated, as recorded in Step C5. Determine number of STR unit of the PCR product(s) of each individual by applying the Distance Migrated by each band (recorded in Step 6) to the Standard Curve plotted. Record in the table below Number of STR Unit Individual ID Larger Band's Number of STR Units Smaller Band's Number of STR Units

11 9. As a group, discuss and answer the following questions: a) Based on the information obtained in the test (or using Figure 1 as an example), which of the individual(s) is heterozygous at the D1S58 locus and which of the individual(s) is homozygous? b) Using the information obtained in the test (or using Figure 1 as an example), suggest and explain two individuals that may be related genetically. c) Using the information obtained in the test (or using Figure 1 as an example), suggest and explain two individuals that may not be related genetically. Technical Assistance NORGEN s Technical Service Department is staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of NORGEN products. If you have any questions or experience any difficulties regarding Norgen s Short Tandam Repeat (D1S58) Detection Kit) or NORGEN products in general, please do not hesitate to contact us. NORGEN customers are a valuable source of information regarding advanced or specialized uses of our products. This information is helpful to other scientists as well as to the researchers at NORGEN. We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques. For technical assistance and more information, please contact our Technical Support Team between the hours of 8:30 and 5:30 (Eastern Standard Time) at (905) or Toll Free at or call one of the NORGEN local distributors ( or through at techsupport@norgenbiotek.com Schmon Parkway, Thorold, ON Canada L2V 4Y6 Phone: (905) Fax: (905) Toll Free in North America: Norgen Biotek Corp. PI