Molecular Analysis of Paternity

Amanda C. Sozer1, Charles M. Kelly1, Daniel B. Demers1

1 Fairfax Identity Laboratories, Fairfax, Virginia
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 14.4
DOI:  10.1002/0471142905.hg1404s16
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The unit covers Variable Numbers of Tandem Repeats (VNTR)based paternity analysis as well as the newer methods relying on PCR to analyze sequencespecific polymorphisms and microsatellite regions. The discussion of data analysis and probability calculations has been expanded to address a number of special circumstances, such as the lack of sample from an alleged father, motherless cases, and more.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Analysis of VNTRs by RFLP Technology
  • Basic Protocol 2: Analysis of Polymorphic Loci by PCR
  • Support Protocol 1: Preparation of Genomic DNA from Whole Blood
  • Support Protocol 2: Interpretation, Statistical Evaluation, and Reporting of DNA Profiles: Data from Mother and Alleged Father
  • Support Protocol 3: Interpretation, Statistical Evaluation, and Reporting of DNA Profiles: Special Paternity Cases
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Analysis of VNTRs by RFLP Technology

  Materials
  • DNA from individuals to be tested (e.g., mother, child, alleged father, and human control; see protocol 3)
  • Restriction enzyme and appropriate buffer
  • 0.6% (w/v) agarose minigel (unit 2.7)
  • 20‐cm, 0.7% to 1.0% analytical agarose gel (unit 2.7)
  • DNA molecular size markers
  • TBE buffer (Life Technologies or appendix 2D)
  • 10 mg/ml ethidium bromide ( appendix 2D; store dark up to 1 year at room temperature)
  • Radiolabeled or nonisotopically labeled DNA probe for VNTR sequence identification
  • 0.2 N NaOH
  • 0.2 M Trisdot;Cl, pH 7.5/2× SSC (see appendix 2D for both recipes)
  • SSC ( appendix 2D)
  • Nylon membrane (e.g., PALL Biodyne)
  • 50°C incubator or water bath
  • Whatman 3MM filter paper
  • X‐ray film (e.g., X‐Omat film; Kodak)
  • Additional reagents and equipment for agarose gel electrophoresis, Southern blotting, and hybridization analysis (unit 2.7), phenol extraction and ethanol precipitation of DNA ( appendix 3B), labeling DNA probes by nick translation or random‐primed synthesis ( appendix 3E), and preparation of biotinylated probes (e.g., CPMB UNIT )

Basic Protocol 2: Analysis of Polymorphic Loci by PCR

  Materials
  • Whole blood samples from individuals to be tested (e.g., mother, child, alleged father, and human control)
  • Cell shocking solution (see recipe)
  • Nuclear lysis buffer (see recipe)
  • 10% (w/v) sodium dodecyl sulfate (SDS; appendix 2D)
  • 20 mg/ml proteinase K (Boehringer Mannheim)
  • Saturated NaCl solution
  • 100% and 70% ethanol, room temperature
  • TE buffer, pH 7.5 ( appendix 2D)
  • 0.6% (w/v) agarose minigel (unit 2.7)
  • DNA concentration standards
  • 10 mg/ml ethidium bromide ( appendix 2D; store dark up to 1 year at room temperature)
  • EDTA Vacutainer tube (purple top)
  • 56°C incubator or water bath
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   AABB. 1997. Parentage Testing Standards Committee Annual Report Summary. American Association of Blood Banks, Arlington, VA.
   Baird, M., Balazs, I., Guisti, A., Miyazaki, L., Nicholas, L., Wexler, K., Kanter, E., Glassberg, J., Allen, F., Rubinstein, P., and Sussman, L. 1986. Allele frequency distribution to two highly polymorphic DNA sequences in three ethnic groups and its application to the determination of paternity. Am. J. Hum. Genet. 29:489‐501.
   Balazs, I., Baird, M., Clyne, M., and Meade, E. 1989. Human population genetic studies of the hypervariable DNA loci. Am. J. Hum. Genet. 44:182‐190.
   Bassam, B.J., Caetano‐Anolles, G., and Gresshoff, P.M. 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Biochem. 196:80‐83.
   Brenner, C.H. 1993. A note on paternity computation in cases lacking a mother. Transfusion 33:51‐54.
   Brenner, C.H. 1997. Symbolic kinship program. Genetics 145:535‐542.
   Brenner, C.H. and Morris, J. 1989. Paternity index calculations in single locus hypervariable probes: Validation and other studies. In Proceedings for the International Symposium on Human Identification Data Acquisitions and Statistical Analysis, pp. 21‐53. Promega Corporation, Madison, WI.
   Budowle, B., Guisti, A.M., Wayne, J.S., Baechtel, F.S., Fourney, R.M., Adams, D.E., and Presley, L.A. 1991. Fixed bin analysis for statistical evaluation of continuous distributions of allelic data from VNTR loci for use in forensic comparisons. Am. J. Hum. Genet. 48:841‐855.
   Budowle, B., Baechtel, F.S., Smerick, J.B., Presley, K.W., Giusti, A.M., Parsons, G., Alevy, M.C., and Chakraborty, R. 1995. D1S80 Population data in African Americans, Caucasians, Southeastern Hispanics, Southwestern Hispanics, and Orientals. J. Forensic Sci. 40:38‐44.
   Davis, L.G., Dibner, M.D., and Battey, J.F. 1986. Basic Methods in Molecular Biology. Elsevier, New York.
   Deka, R., DeCroo, S., Yu, L.M., and Ferrell, R.E. 1992a. Variable number of tandem repeat (VNTR) polymorphism at locus D17S5 (YNZ22) in four ethnically defined human populations. Hum. Genet. 90:86‐90.
   Deka, R., Chakraborty, R., DeCroo, S., Rothhammer, F., Barton, S.A., nad Ferrell, R.E. 1992b. Characteristics of polymorphism at a VNTR locus 3′ to the apolipoprotein B gene in five human populations. Am. J. Hum. Genet. 51:1325‐1333.
   Dieffenbach, C.W. and Dveksler, G.S. 1993. Setting up a PCR Laboratory. In PCR Methods and Applications, 3:S2‐S7. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Endean, D. 1989. RFLP analysis and paternity testing: Observations and caveats. In Proceedings for the International Symposium on Human Identification Data Acquisition and Statistical Analysis, pp. 55‐75. Promega Corporation, Madison, WI.
   Endean, D. 1995. Apparent false exclusion observed in various DNA‐VNTR systems reported by AABB‐accredited parentage testing laboratories. In American Association of Blood Banks, Accreditation Requirements Manual, 2nd ed., pp. 66. American Association of Blood Banks, Arlington, VA.
   Evett, I.W., Werrett, D.J., Gill, P., and Buckelton, J.S. 1989a. DNA fingerprinting on trial. Nature (Lond.). 40:435.
   Evett, I.W., Werrett, D.J., and Smith, A.F. 1989b. Probabilistic analysis of DNA profiles. J. Forensic Sci. Soc. 29:191‐196.
   Garber, R.A. and Morris, J.W. 1983. General equations for the average power of exclusion for genetic systems of n codominant alleles in one‐parent and no‐parent cases of disputed parentage. In Inclusion Probabilities in Parentage Testing (R.H. Walker, ed.). American Association of Blood Banks, Arlington, VA.
   Gene, M., Huguet, E., Sanchez‐Garcia, C., Moreno, P., Corbella, J., and Mezquita, J. 1995. Study of the 3′‐Apo B minisatellite performed in the population of Catalonia (Northeast Spain). Hum. Hered. 45:70‐74.
   Gjertson, D.W. and Endean, D. 1996. Promega Statistics Workshop, Scottsdale, AZ.
   Gjertson, D.W., Mickey, M.R., Hopfield, J., Takenouchi, T., and Terasaki, P.I. 1988. Calculation of the probability of paternity using DNA sequences. Am. J. Hum. Genet. 43:860‐869.
   Henke, J., Fimmers, R., Baur, M.P., and Henke, L. 1993. DNA minisatellite mutations: Recent investigations concerning distribution and impact on parentage testing. Int. J. Leg. Med. 105:217‐222.
   Herrin, G., Jr. 1992. A comparison of models used for RFLP pattern frequencies. J. Forensic Sci. 37:1640‐1651.
   Herrin, G., Jr., Forman, L., and Garner, D.D. 1990. The use of Jeffrey's multilocus and single locus probes in forensic analysis. In DNA and Other Polymorphisms in Forensic Science (H.C. Lee and R.E. Gaensslen, eds.) pp. 45‐60. Yearbook Medical, Chicago, Ill.
   Hou, Y., Schmitt, C., Staak, M., Puers, C., and Prinz, M. 1994. Genetic variation of the amplified VNTR polymorphism COL2A1 in Chinese and German populations. Hum. Hered. 44:114‐119.
   Jenkins, F.J. 1994. Basic methods for the detection of PCR products. PCR. Methods Appl. 3:S77‐S82.
   Kirby, L.T. 1990. DNA Fingerprinting: An Introduction. Stockton Press, New York.
   Lander, E.S. 1989. DNA fingerprinting on trial. Nature 339:501‐505.
   McKusick, V.A., Ferrara, P.B., Kazazizn, H.H., King, M.C., Lander, E.S., Lee, H.C., Lempert, R.O., Macklin, R., and Marr, T. 1992. DNA Technology in Forensic Science. National Academy Press, Washington, D.C.
   Morris, J. 1992. A review of DNA analysis in paternity testing. In Proceedings from the Third International Symposium on Human Identification: Application of New Technologies, Standardization and Validation of Methods, Data Sharing, and Legal Issues Affecting the DNA Typing Community, pp. 177‐189. Promega Corporation, Madison, Wis.
   Morris, J. 1993. Application of genetic testing to disputed paternity. Clin. Chem. 39:716‐717.
   Morris, J.W. and Brenner, C.H. 1991. Paternity index calculations in single locus hypervariable DNA probes: Further validation studies. In Proceedings from Second International Symposium on Human Identification: New Technologies, Standardization of Methods, and Data Sharing for DNA Typing Laboratories, pp. 181‐190. Promega Corporation, Madison, Wis.
   Morris, J.W., Sanda, A.I., and Glassberg, J. 1989. Biostatistical evaluation from continuous allele frequency distribution deoxyribonucleic acid (DNA) probes in reference to disputed paternity and identity. J. Forensic Sci. 34:1311‐1317.
   Nakamura, Y., Leppert, M., O'Connell, P., Wolff, R., Holm, T., Culver, M., Martin, C., Fujimoto, E., Hoff, M., Kumlin, E., and White, R. 1987. Variable number of tandem repeats (markers) for human gene mapping. Science 235:1616‐1622.
   Parentage Testing Accreditation Requirements Manual, 2nd ed., 1995. American Association of Blood Banks, Bethesda, Md.
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, 2nd ed. Cold Spring Harbor, N.Y.
   Schneeberger, C., Speiser, P., Kury, F., and Zeillinger, R. 1995. Quantitative detection of reverse transcriptase–PCR products by means of a novel and sensitive DNA stain. PCR Methods Appl. 4:234‐238.
   Sharp, P.A., Sugden, B., and Sambrook, J. 1973. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose. Biochemistry. 12:3055‐3063.
   Shields, W.M. 1992. Forensic DNA typing as evidence in criminal proceedings: Some problems and potential solutions. In Proceedings from Third International Symposium on Human Identification: Application of New Technologies, Standardization and Validation of Methods, Data Sharing, and Legal Issues Affecting the DNA Typing Community, pp. 1‐50. Promega Corporation, Madison, Wis.
   Silver, H. 1982. Probability of Inclusion in Paternity Testing. American Association of Blood Banks, Bethesda, Md.
   Svensmark, O. and Eriksen, B. 1991. Measurement errors in DNA‐profiling. In Proceedings from Second International Symposium on Human Identification: New Technologies, Standardization of Methods, and Data Sharing for DNA Typing Laboratories, p. 322 Promega Corporation, Madison, Wis.
   Technical Working Group on DNA Analysis Methods (TWGDAM). 1995. Guidelines for a quality assurance program for DNA analysis. Federal Bureau of Investigation, Quantico, VA.
   Traver, M. 1996. Promega Statistics Workshop, Scottsdale, AZ.
   Walker, R.H. 1983. Inclusion Probabilities in Parentage Testing. American Association of Blood Banks, Bethesda, Md.
   Wyman, A. and White, R. 1980. A highly polymorphic locus in human DNA. Proc. Natl. Acad. Sci. U.S.A. 77:6754‐6758.
Key Reference
   Allen, R.W., Wallhermfechtel, M., and Miller, W.V. 1990. The application of restriction fragment length polymorphism mapping to parentage testing. Transfusion 30:552‐564.
  Summarized all RFLP methods and was responsible for creating methodology and initial testing for DNA and parentage.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library