Publication No. 10765
DNA Paternity Testing
Student Laboratory Kit
Materials Included In Kit
Agarose, powder, electrophoresis grade, 3 g
Additional Materials Required
TAE Electrophoresis buffer, concentrate 50X, 20 mL*
Preparation of 1X Electrophoresis Buffer
Note: Prepare enough buffer solution to allow each group to cover the gel in the chamber to a depth of about 2 mm. Depending on the type of electrophoresis units being used, the amount of buffer needed may be as much as 300 mL per chamber. Gel preparation requires an additional 60 mL of buffer to make a 6 x 6 cm gel.
Preparation of 1X Methylene Blue Electrophoresis Stain
Note: 40 mL is enough to stain a gel in the staining tray that is provided.
Electrical Hazard: Treat these units like any other electrical source—very carefully! Be sure all connecting wires, terminals and work surfaces are dry before using the electrophoresis units. Do not open the lid of the unit while the power is on. Exercise extreme caution in handling the methylene blue—it will readily stain clothing and skin. Wearing chemical splash goggles and gloves is strongly recommended. Wash hands thoroughly with soap and water before leaving the laboratory. Please consult current Safety Data Sheets for additional safety, handling and disposal information.
Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All solutions used in this lab may be disposed of down the drain using copious amounts of water according to Flinn Suggested Disposal Method #26b. Used gels may be disposed of in the regular trash according to Flinn Suggested Disposal Method #26a. The DNA in this kit is derived from bacteriophage samples. It is not pathogenic to humans and therefore it is not considered a biohazard.
Correlation to Next Generation Science Standards (NGSS)†
Science & Engineering PracticesPlanning and carrying out investigations
Analyzing and interpreting data
Developing and using models
Constructing explanations and designing solutions
Engaging in argument from evidence
Disciplinary Core IdeasMS-LS3.A: Inheritance of Traits
MS-LS1.A: Structure and Function
HS-LS3.A: Inheritance of Traits
HS-LS1.A: Structure and Function
Crosscutting ConceptsCause and effect
Structure and function
MS-LS3-2: Develop and use a model to describe why asexual reproduction results in offspring with identical genetic information and sexual reproduction results in offspring with genetic variation.
DNA Banding Worksheet
The banding patterns will vary due to differences in the electrophoresis run and the periodic substitution of DNA. All DNA will be between 75 and 24,000 bp.
Migration distances will vary due to differences in the electrophoresis run and the periodic substitution of DNA. All DNA will be between 75 and 24,000 bp.
Data Table 1
Answers to Questions
Blood typing cannot exclude a father with the same blood type from paternity. HLA cannot differentiate between related alleged father.
Colored tracking dyes are used to visualize the position of the colorless DNA within the gel during electrophoresis.
The smallest fragments will have moved the farthest distance away from the sample wells during electrophoresis.
PCR testing should be used on the degraded, small quantities of DNA.
Samples are mixed with a restriction enzyme and tracking dyes, loaded into the wells on a submerged agarose gel inside a gel electrophoresis chamber. Current is applied to the system and the DNA is separated according to their fragment length. The electrophoresis is stopped and the samples are transferred to a Southern blot membrane and the radioactive labels attach to the DNA fragments. The radioactive DNA is placed on a sheet of X-ray film and the resulting autorad is analyzed for matching DNA bands.
a. Not placing the sample deep enough into the well or not placing enough sample into the well.
DNA sample 3 and DNA sample 4 are a match.
DNA Paternity Testing
The question of whether or not a child is biologically related to a probable father has been around for many years. Prior to the 1920s, paternity questions were resolved by observing the child’s phenotype—does the child physically resemble the alleged father? This method was not accurate since genetic recombination that occurs during the formation of gametes, results in a unique set of genes and typically, a blend of parental features. Recently, paternity testing has become a very reliable, high tech procedure with the development of DNA testing for relatedness.
The first laboratory testing for paternity involved blood typing. Proteins found on the surface of red blood cells determine whether someone has blood type A, B, AB or O. Two proteins, called A and B, are coded on chromosome 9. Blood typing was only able to exclude someone as a father if the child and father had different dominant blood types. For example, a father with a type B blood and a mother with a type O blood could not have a child with the type A or AB blood. If the child was found to have the type B blood, the tested father could not be excluded from paternity along with any other type B male. As is often the case in biology, it was later discovered that inheritance of blood type is not as simple as it was first thought. Inheritance of A, B and AB blood types are confounded by the presence of a third protein, type H, which when inherited in the homozygous recessive form (hh) can block the production of the A and B proteins. This leads to a type O phenotype even if the parents both have type A or type B blood. As a consequence of this “Bombay phenotype,” blood typing is no longer considered an accurate method for determining paternity.
In order to test a person’s VNTR profile, long strands of DNA are extracted from cells collected from the mother, child and alleged father. The strands of DNA are cut into fragments using special enzymes called restriction enzymes. The enzymes that break DNA molecules at internal positions are called restriction endonucleases. Enzymes that degrade DNA by digesting the molecule from the ends of the DNA strand are termed exonucleases. There are several different restriction enzymes available to molecular biologists. Each restriction enzyme recognizes a specific nucleotide sequence. The enzyme “scans” the length of the DNA molecule and then digests it (breaks it apart) at or near a particular recognition sequence. The specific sequence may be five to sixteen base pairs long. For example, the HindIII endonuclease has the following six-base-pair recognition sequence:
It breaks the DNA at the locations indicated by the dotted line and produces jagged ends, which molecular biologists call sticky ends. Other endonucleases cut the DNA cleanly at one specific base-pair and produce blunt ends.
The general procedure for DNA analysis is as follows. The RFLP fragments generated by the restriction enzyme are loaded into wells made in an agarose gel. Agarose is a refined form of agar. The agarose gel is positioned between two electrodes with the wells toward the cathode (negative electrode). When a voltage is applied to the electrondes, the negatively charged DNA fragments move toward the anode (positive electrode) (see Figure 1). The electrophoresis chamber is filled with a buffer solution, bathing the gel in a solution that shields the system from changes in pH. The gel acts like a molecular sieve, creating a maze for the fragments to move through on their way toward the anode. Smaller fragments move faster through the holes or pores in the gel, while larger fragments move slower because of their size.
Paternity testing is just one type of relatedness that can be tested. Tests can determine if a person is related to a mother, father, sister, brother or grandparent. In this lab, the relationship between descendants of one man will be determined. See the included scenario handout (BAP10765B). The purpose of this activity is to demonstrate the separation technique known as gel electrophoresis.
Electrical Hazard: Treat these units like any other electrical source—very carefully! Be sure all connecting wires, terminals and work surfaces are dry before using the electrophoresis units. Do not open the lid of the unit while the power is on. Use heat protective gloves and eye protection when handling hot liquids. Methylene blue will stain skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.
Part A. Loading a Gel
Part B. Running a Gel
Part C. Staining the DNA
Part D. Storing the Gel