[ GeneKin^® Y-STR Systems ]

Contents

Description
  A. GeneKin^® Y-STR systems (Silver Stain Detection)
  B. Advantages of GeneKin^®  Y-STR Systems typing
 
GeneKin^® Y-STR systems (Silver Stain Detection)

Amplification
  A. Choice of Thermal Cycling Protocol
  B. Amplification Set-Up
  C. Amplification Thermal Cycling
  D. Agarose Gel Electrophoresis of Amplification Products (optional)

Electrophoresis and Detection
 Polyacrylamide Gel Preparation
  A. Notes
  B. Procedure

 Polyacrylamide Gel Electrophoresis
  A. Gel Pre-Run
  B. Sample Preparation
  C. Sample Loading
  D. Gel Electrophoresis

Silver Staining
  A. Notes
  B. Procedure

Results

References

Description

A. GeneKin^® Y-STR systems (Silver Stain Detection)

  Interspersed short tandem repeat (STR) loci consist of tandem arrays raging from 1 to 6 base pairs in length (1-4). These STR sequences represent a significant fraction of eukaryote (e.g., human) genomes and are detected mostly by polymerase chain reaction (PCR) analysis (5-8). Alleles of these loci are differentiated by the number of copies of the repeat sequence contained within the amplified region and are distinguished from one another using silver stain following electrophoretic separation.

  The usefulness of Y-chromosome markers for studies of human population genetics and forensic/paternity analysis has recently  been recognized (9-10). The human Y-chromosome of the non-recombining portions has special features: a haploid transmission pattern; and father-to-son transmission. The DNA sequence of the non-recombining portions contains a genetic record only of the mutational events that occurred in their past (11-17).

  GeneKin^® Y-STR Systems are more useful for investigating and reconstructing the male mediated forensic/paternity testing (18). Moreover, GeneKin^® Y-STR Systems will be of great benefit in analyzing mixed DNA samples, in investigating sexual assaults, as well as in paternity testing (paternal kinship test) where the alleged father is not available but other patrilineal relatives are (19).

B. Advantages of GeneKin^® Y-STR Systems typing

  The GeneKin Y-STR Systems typing provide a rapid, non-radioactive method, which can be used to evaluate small amounts (e.g., 50 ng) of human DNA. The protocols detailed in this manual describe the use of silver staining (20) to detect the presence of amplified STR products following their separation by denaturing polyacrylamide gel electrophoresis.

  In addition to these advantages, the Y-STR loci chosen for inclusion in the GeneKin^® Y-STR Systems contain alleles of discrete and separable lengths. This allows the construction of allelic ladders, which contain fragments of the same lengths as several or all known alleles for the locus. Visual comparison between the allelic ladder and amplified samples of the same locus allows rapid and precise assignment of alleles.

 
GeneKin^® Y-STR systems (Silver Stain Detection)

  The GeneKin^® Y-STR Systems provide all of the component required to perform 100 amplification reactions except Taq DNA polymerase and sample DNA.

Storage Conditions: Store all components at -20¡É. The pre- and post-amplification components (Allelic Ladder Mix, Loading Solution and Control DNA) are sealed in separate packages to prevent contamination. These systems are guaranteed for at least 6 months from date of purchase.

Table 1. GeneKin^®  Y-STR Systems Information^*

^* Korean data
^** In Y-STR systems, haplotype diversity value is the same as the discrimination index.

Amplification

  The GeneKin^® Y-STR Systems have been developed for amplification without artifacts using standard Taq DNA polymerase. Special enzymes such as AmpliTaq Gold  are not required for peak performance.

Materials to Be Supplied by the User

¡€ Thermal cycler, model PTC-200 (MJR)/ GeneAmp system 9700 (Perkin-Elmer) or GeneAmp  system 9600 (Perkin-Elmer)
¡€ Microcentrifuge
¡€ Taq DNA polymerase
¡€ Sterile water
¡€ Mineral oil
¡€ 0.5 §¢ or 0.2 §¢ microcentrifuge tubes (compatible with thermal cycler)
¡€ 1.5 §¢ microcentrifuge tubes
¡€ Autoclaved tips
¡€ Ice

A. Choice of Thermal Cycling Protocol

  The GeneKin^® Y-STR systems and their corresponding singleplexes are optimized for use with  PTC-200 (MJR)/ GeneAmp 9700 thermal cycler and GeneAmp 9600 thermal cycler. The GeneKin^® Y-STR systems were optimized for use with 0.2 §¢ PCR tube and the PTC-150 (MJR) thermal cycler. However, each system may be used with their thermal cycler.

Please refer to Table 2 and 3 for recommended protocol for multiplex systems and singleplex systems, respectively.

  When using a thermal cycler on which a system was not optimized, there may be a small loss in product yield or sensitivity, and the balance between loci may change slightly in multiplex systems, Meticulous care must be taken to ensure successful amplification.

Table 2. A list of the PCR cycling conditions of Multiplex GeneKin^® Y-STR systems

Table 3. A list of the PCR cycling conditions of Singleplex GeneKin^® Y-STR systems

B. Amplification Set-Up

The use of gloves, autoclaved pipet tips and microcentrifuge tubes is highly recommended to prevent contamination.

  1. Thaw the Y-STR 10¡¿ Buffer and Y-STR 10¡¿ Primer Pair(s) and then keep on ice.
  2. Place one clean, autoclaved 0.5 §¢ microcentrifuge tube for each reaction into a rack and label appropriately.
  3. Determine the number of reaction to be set up. This should include a positive and negative control reaction. Add 1 or 2 reactions to this number to compensate for pipetting error. While this approach does waste a small amount of each reagent, it ensures that you will have enough PCR master mix for all samples.
  4. Calculate the required amount of each component of the PCR master mix Multiply the volume (§¡) per sample by the total number of reaction (from step 3) to obtain the final volume (§¡).
  5. In the order listed in Table 4, add the final volume of each reagent to a sterile tube. Mix gently (do not vortex) and place on ice.
  6. Add 22.5 §¡ of PCR master mix to each tube and place on ice.

Table 4. PCR Amplification Reaction Set-Up of the GeneKin^® Y-STR systems.

  7. Pipet 2.5 §¡ of each sample into the respective tube containing 22.5 §¡ of PCR Master Mix.

  Notes:
  For the GeneKin^® Y-STR systems¥±, ¥² and ¥³ use 25 ng of template DNA.

  For the GeneKin^® Y-STR systems¥°, use 25-50 ng highly purified template DNA. A highly qualified template DNAs should be required in the GeneKin^® Y-STR systems¥° (e.g., Promega Wizard DNA clean-up system; Cat # A7280 or QIAGEN QIAamp DNA mini kit; Cat # 51304).

  8. Pipet 2.5 §¡ (25 ng) of control DNA (diluted to 10 ng/§¡) into a 0.5 §¢ microcentrifuge tube. containing 22.5 §¡ of PCR master mix, as a positive amplification control.
  9. Pipet 2.5 §¡ of sterile water (instead of template DNA) in to a 0.5 §¢ microcentrifuge tube, containing 22.5 §¡ of PCR master mix, as a negative amplification control.
  10. if recommended by the cycling protocol, add 1 drop of mineral oil to each tube. Close the tubes.
  11. Centrifuge the samples briefly to bring the contents to the bottom of the tube.

C. Amplification Thermal Cycling
  1. Assemble the Tubes in a thermal cycler.
  2. Select and run a recommended protocol from Table 4.
  3. After completion of the thermal cycling protocol, store the samples at -20¡É.

  Note: Storage of amplified samples at 4¡É or higher may produce degradation products.

D. Agarose Gel Electrophoresis of Amplification Products (optional)
  This procedure is optional if PCR is routinely performed in your laboratory. You may use agarose gel electrophoresis to confirm the success and product yield of the amplification reaction quickly before performing polyacrylamide gel electrophoresis.

Materials to Be Supplied by the User

1X TAE buffer (pH 7.9)
Agarose (e.g. FMC Seakem GTG grade)
6X loading solution
10 §·/§¢ ethidium bromide stock solution

  1. Prepare a 2% agarose gel by adding 2.0 g of agarose to 100 §¡ of 1X TAE buffer. Mark the liquid level in the container, then boil or heat in a microwave oven to dissolve the agarose. Add preheated deionized water to make up for any volume lost due to evaporation.
  2. Cool the agarose to 55¡É before pouring into the gel tray. Be sure that the gel tray is level. Pour the agarose into the tray, insert the gel comb and allow to set for 20-30 minutes.
  3. Prepare the samples by mixing 10 §¡ of each sample with 2.0 §¡ of 6X loading solution.
  4. Prepare 1 liter of 1X TAE for the electrophoresis running buffer.
  5. Place the gel and tray in the electrophoresis gel box. Pour the running buffer into the tank. The buffer should cover the gel to a depth of at least 0.65 §¯, the thickness of the gel.  Gently remove the comb.
  6. Load each sample mixed with 6X loading solution (see Step 3).
  7. Set the voltage at 100 volts (measured as the distance between the two electrodes). Allow the gel to run for 30 minute.
  8. After electrophoresis, stain the gel in 1X TAE containing 0.5 §·/§¢ ethidium bromide. Gently rock for 10 minutes at room     temperature. Remove the ethidium bromide solution and replace with deionized water. Allow the gel to destain for 10 minutes.
  9. Using a UV transilluminator (302 nm), photograph the gel.

  Note: When analyzing the data, do not be alarmed if you see extra bands in addition to the alleles. DNA heteroduplexes can be expected when performing nondenaturing agarose gel electrophoresis. The sole purpose of the agarose gel is to confirm the success of the PCR reaction.

Electrophoresis and Detection

Polyacrylamide Gel Preparation

Materials to Be Supplied by the User

¡€ Deionized Water

¡€ 6% denatured polyacrylamide gel
¡€ 10X TBE Buffer
¡€ 10% Ammonium Persulfate
¡€ TEMED
¡€ Nalgene  tissue culture filter (0.45 §­)
¡€ polyacrylamide gel electrophoresis apparatus for gels ¡Ã30 §¯ (e.g. BRL SA32, BRL S2, OWL ADJ1, Bio-Rad Sequi-Gen GT Sequencing Cell)
¡€ glass plates and side spacers for polyacrylamide gel ¡Ã30 §¯
¡€ comb (18 wells: 0.8 §® thick, Gibco BRL; Cat.# 11092-103, 34-60 wells: 0.4-0.8 §® thick, Owl Scientific; Cat.# S2S-60A; 34 wells: 0.75   §® thick, Bio-Rad; Cat.# 165-3858)
¡€ power supply
¡€ clamps (e.g., large office binder clips)
¡€ diamond pencil for marking glass plates

A. Notes
  1. Use 6% acrylamide for GeneKin^® Y-STR systems.
  2. Unpolymerized acrylamide is a neurotoxin and suspected carcinogen; avoid inhalation and contact with skin. Read the warning label and take the necessary precautions when handling this substance. Always wear two pairs of gloves and safety glasses when working with acrylamide powder or solutions.
  3. All cleaning utensils (sponges) for the longer glass plates should be kept separate from those for the shorter glass plates to prevent cross-contamination of the binding solution.
  4. New glass plates should be soaked in 2M NaOH for 1 hour and then rinsed thoroughly with deionized water before use. New plates also should be etched with a diamond pencil in the corner of one side to distinguish the sides of the plates in contact with the gel.

B. Procedure
  The following protocol is for the preparation of a denaturing polyacrylamide gel with the dimensions of 21.0 §¯ wide x 40.0 §¯ high x 0.75 §® thick (e.g., the Sequi-Gen GT sequencing gel electrophoresis apparatus, Bio-Rad Cat.# 165-3860).

  1. Thoroughly clean the shorter and longer glass plates once with sterilized water, twice with 95% ethanol and Kimwipes systems tissues.
  2. Assemble the glass plates by placing 0.75 §® side spacers (Bio-Rad Cat.# 165-3819) between the plates and using clamps to hold them in place. Lean the assembled plates against a test tube rack or other similar support.
  3. Prepare a 6% acrylamide solution by adding together the ingredients listed below.

  4. Add 12 §¡ of TEMED and 1 §¢ of 10% ammonium persulfate to the 60 §¢ of 6% acrylamide solution and mix gently.
  5. Carefully pour the acrylamide solution between the glass plates. To prevent bubble formation, start pouring at one side of the assembled plates and maintain a constant flow of solution.
  6. Position the gel horizontally, resting it on two test tube racks or other similar supports. Remove any bubbles that may have formed.
  7. Insert combs straight side into the gel, between the glass plates until the teeth are almost completely inserted into the gel.
  8. Secure the comb(s) with 2-3 clamps each.
  9. Pour the remaining acrylamide solution into a disposable conical tube as a polymerization control. Rinse the squeeze bottle, including the spout, with water.
  10. Allow polymerization to proceed for at least 1 hour. Check the polymerization control to be sure that polymerization has occurred.

  Note: The gel may be stored overnight if a paper towel saturated with deionized water and plastic wrap are placed around the well end of the gel to prevent the gel from drying out (crystallization of the urea will destroy the gel). If no bottom spacer is used, the bottom of the gel should be wrapped.

Polyacrylamide Gel Electrophoresis

A. Gel Pre-Run
  1. Remove the clamps from the polymerized acrylamide gel and clean the glass plates with paper towels saturated with deionized water.
  2. Shave any excess polyacrylamide away from the comb. Remove the comb.
  3. Add 1X TBE to the bottom chamber of the electrophoresis apparatus.
  4. Gently lower the gel (glass plates) into the buffer with the longer plate facing out and the well-side on top.
  5. Secure the glass plates to the sequencing gel apparatus.
  6. Add 1X TBE to the top buffer chamber of the electrophoresis apparatus.
  7. Using a syringe filled with buffer, remove the air bubbles on the top of the gel. Be certain the well area is devoid of air bubbles and small pieces of polyacrylamide. Use a syringe with a bent 19-gauge needle to remove the air bubbles between the glass plates on the bottom of the gel.
  8. Pre-run the gel to achieve a gel surface temperature of approximately 50¡É. Consult the manufacturer's instruction manual for the recommended electrophoresis conditions.

  Note: As a reference, we generally use 55 - 60 Watts for a 40 §¯ polyacrylamide gel, 50 - 55 Watts for a 32 §¯ gel. The gel running conditions may have to be adjusted in order to reach a temperature of 50¡É.

B. Sample Preparation
  1. Prepare the PCR samples by mixing 2.5 §¡ of each sample with 2.5 §¡ of STR 2X Loading Solution.

Note: The sample alleles may appear more intense than ladder alleles on the gel, but this should not interfere with allele determination. For more even band intensities, mix 1 §¡ of each sample with 4 §¡ of a pre-mix of 2.5 §¡ STR 2X Loading Solution + 1.5 §¡ STR 1X Buffer or mix 5 §¡ sample with 1 §¡ of 6¡¿ Loading Solution (not provided).

  2. Add 2.5 §¡ of the Y-STR Ladder to 2.5 §¡ of STR 2X Loading Solution for each ladder lane. The number of ladder lanes used depends on personal preference.
  3. Briefly spin the samples in a microcentrifuge to bring the contents to the bottom of the tube.

C. Sample Loading
  1. Denature the samples by heating at 98¡É for 5 minutes and immediately chill on ice.

Note: Denature the samples just prior to loading the gel. Sample DNA will re-anneal if denatured hours before loading. This may produce fragments of indeterminate migration.

  2. After the pre-run, use a 50-100 cc syringe filled with buffer to flush the urea from the well area.
  3. Load 3 §¡ of each sample into the respective wells. The loading process should take no longer than 20 minutes to prevent the gel from cooling.

D. Gel Electrophoresis
  1. At the completion of loading, run the gel using the same conditions as in Section Gel Pre-Run.

Note: In a 6% gel, bromophenol blue migrates at approximately 25 bases and xylene cyanol migrates at approximately 105 bases.

  2. Knowing the size ranges for each locus (see Tables 1) and migration characteristics of the dyes (see Step 1, above), stop electrophoresis any time after the locus of interest has passed the midpoint of the gel. If running more than one locus or a multiplex, be careful not to run the smallest locus off the bottom of the gel. The gel bands may be detected by silver staining.

Silver Staining
This protocol describes the use of modified protocol in Promega's DNA Silver Staining System (Cat.# DQ7050).

Materials to Be Supplied by the User

¡€ Deionized Water

¡€ 1§€ fix/stop solution (10% glacial acetic acid)
¡€ 500 §¢ staining solution (0.5 g Silver Nitrate, 750 §¡ 37£¥ Formaldehyde)
¡€ 500 §¢ developer solution (15 g Sodium Carbonate, 750 §¡ 37£¥ Formaldehyde, 100 §¡ Sodium Thiosulfate)
¡€ Heating block, 100¡É
¡€ Glass dish (PYREX, 23 ¡¿ 35 ¡¿7 §¯)
¡€ Orbital shaker or rocker platform
¡€ Illuminator

A. notes
  1. Steps involving solutions containing formaldehyde should be performed in a chemical hood.
  2. Chill the developer solution to 4-10¡É.
  3. Use 500 §¢ of each solution per gel for each step (for a 23 ¡¿ 35 x 7 §¯ dish).
  4. Gently agitate during each step.
  5. Use fingers, gently touch the gel and pouring over each solution on to the gel.
  6. The duration of "Step 4e" of below Procedure Section is important. The total time from immersion in deionized water to immersion in developer solution should be less than 10 seconds. If the deionized water rinse step does exceed 10 seconds, repeat Step 4d of below Procedure Section.

B. Procedure
  1. After electrophoresis, empty the buffer chambers and carefully loosen the gel clamps. Remove the glass plates from the apparatus.
  2. Place the gel (glass plates) on a flat surface. Remove the side spacers. Use a plastic wedge to carefully separate the two glass plates.
  3. Place the gel in a shallow glass dish by flow the fixer to gel attached plate.
  4. To silver stain, follow Steps a-h:

  5. Observe the gel on the illuminator.
  6. Attach the Gel on a 3MM chromatography paper or acetate film.
  7. Dry the gel using gel dryer.

Results

Figure 1. Electrophoretic separation of the DYS392/DYS19/DYS388 PCR fragments in a denaturing polyacrylamide gel and silver detection using the GeneKin^® Y-STR Systems¥°. The results of DNA profile indicate that non-paternity is suspected (II-3).

Figure 2. Electrophoretic separation of the DYS391/DYS390/DYS393 PCR fragments in a denaturing polyacrylamide gel and silver detection using the GeneKin^® Y-STR Systems¥±.The results of DNA profile indicate that non-paternity is suspected (II-3).

Figure 3. Electrophoretic separation of the DYS389¥±/DYS389¥°/DXYS156XY PCR fragments in a denaturing polyacrylamide gel and silver detection using the GeneKin^® Y-STR Systems¥².The results of DNA profile indicate that non-paternity is suspected (II-3).

Figure 4. Electrophoretic separation of the DYS385/DXYS156XY PCR fragments in a denaturing polyacrylamide gel and silver detection using the GeneKin^® Y-STR Systems¥³. The results of DNA profile indicate that non-paternity is suspected (II-3).

References

1. Edwards, A. et al. (1991) DNA typing with trimeric and tetrameric tandem repeats: polymorphic loci, detection systems, and population genetics. In: The Second International Symposium on Human Identification 1991, Promega Corporation, 31.

2. Edwards, A. et al. (1991)  DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. Am. J. Hum. Genet. 49, 746.

3. Edwards, A. et al. (1992) Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics 12, 241.

4. Jeffrey, A.J., Wilson, V. and Thein, S.L. (1985) Hypervariable ¡°minisatellite¡±regions in human DNA. Nature 314, 67-73.

5. Ausubel, F.M. et al. (1993) Unit 15: the polymerase chain reaction. In: Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, NY.

6. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Chapter 14: in vitro amplification of DNA by the polymerase phain reaction. In: Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

7. PCR Technology: Principles and Applications for DNA Amplification (1989), ed., Erlich, H.A., Stockton Press, NY.

8. PCR Protocols: A Guide to Methods and Applications (1990) eds., Innis, M.A. et al. Academic Press, San Diego, CA.

9. Kim, Y.J., Shin, D.J., Kim, J.M., Jin, H.J., Kwak, K.D., Han, M.S., Choi, S.K. and Kim, W. (2001) Y-chromosome STR haplotype profiling in the Korean population. Forensic Sci. Int. 115, 231-237.

10. Roewer, L., Kayser, M., de Knijff, P., Anslinger, K., Betz, A., Caglia, A., Corach, D., Furedi, S., Henke, L., Hidding, M., Kargel, HJ., Lessig, R., Nagy, M., Pascali, V.L., Parson, W., Rolf, B., Schmitt, C., Szibor, R., Teifel-Greding, J. and Krawczak, M. (2000) A new method for the evaluation of matches in non-recombining genomes: application to Y-chromosomal short tandem repeat (STR) haplotypes in European males. Forensic Sci Int. 114: 31-43.

11. Hammer, M.F. (1995) A recent common ancestry for human Y chromosomes. Nature 378,  376-378.

12. Jobling, M.A. and Tyler-Smith, C. (1995) Fathers and sons: the Y chromosome and human evolution. Trends Genet. 11, 449-456.

13. Edwards, A., Hammond, H.A., Jin, L., Caskey, C.T. and Chakraborty, R. (1992) Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics 12, 241-253.

14. Hammond, H.A., Jin, L., Zhong, Y., Caskey, C.T. and Chakraborty, R. (1994) Evaluation of 13 short tandem repeat loci for use in personal identification applications. Am. J. Hum. Genet. 55, 175-189.

15. de Knijff, P., Kayser, M., Caglia, A., Corach, D., Fretwell, N., Gehrig, C., Graziosi, G., Heidorn, F., Herrmann, S., Herzog, B., Hidding, M., Honda, K., Jobling, M., Krawczak, M., Leim, K., Meuser, S., Meyer, E., Oesterreich, W., Pandya, A., Parson, W., Penacino, G., Perez-Lezaun, A., Piccinini, A., Prinz, M., Schmitt, C., Schneider, P.M., Szibor, R., Teifel-Greding, J., Weichhold, G. and Roewer, L. (1997) Chromosome Y microsatellites: population genetic and evolutionary aspects. Int. J. Legal Med. 110, 134-140.

16. Kayser, M., Caglia, A., Corach, D., Fretwell, N., Gehrig, C., Graziosi, G., Heidorn, F., Herrmann, S., Herzog, B., Hidding, M., Honda, K., Jobling, M., Krawczak, M., Leim, K., Meuser, S., Meyer, E., Oesterreich, W., Pandya, A., Parson, W., Penacino, G., Perez-Lezaun, A., Piccinini, A., Prinz, M., Schmitt, C., Schneider, P.M., Szibor, R., Teifel-Greding, J., Weichhold, G., de Knijff, P. and Roewer, L. (1997) Evaluation of Y-chromosomal STRs: a multicenter study. Int. J. Legal Med. 110, 125-133.

17. Puers, C. et al. (1994) Analysis of polymorphic STR loci using well-characterized allelic ladders. In: Proceedings from the Fourth International Symposium on Human Identification 1993, Promega Corporation, 161.

18. Shin, D.J., Jin, H.J., Kwak, K,D., Choi, J.W., Han, M.S., Kang, P.W., Choi, S.K. and Kim W., (2001) Y-Chromosome Multiplexes and Their Potential for the DNA Profiling of Koreans. Int. J. Legal Med. 115: 109-117.

19. Shin, D.J., Kim, J.M., Jin, H.J., Kwak, K.D., Han, M.S., Kang, P.W., Choi, S.K. and Kim, W. (2000) Y-chromosome STRs typing and population study for the DNA Profiling in Koreans. Korean J. Forensic Sci. 12: 267-273.

20. Bassam, B.J., Caetano-Anolles, G. and Gresshoff, P.M. (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Biochem. 196, 80