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Keywords:

  • PCR;
  • DNA extraction;
  • DNA gel electrophoresis;
  • Alu repeat;
  • Hardy-Weinberg statistics

Abstract

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS AND DISCUSSION
  5. SAFETY ISSUES
  6. RELEVANT WEBSITES
  7. REFERENCES

A 3-week undergraduate molecular biology laboratory exercise combining commercially available kits is described. The laboratory involves DNA extraction from a human dried blood sample, amplification of an Alu-repeat sequence found on chromosome 8, and analysis of the genetic and allelic frequency in the class population using Hardy-Weinberg statistics. The use of the prepared kits provides a means for the instructor of an undergraduate biochemistry course to explore molecular biology techniques on a limited budget and with a minimal time investment.

The techniques used in a biochemistry laboratory are constantly changing as new methodologies are developed by industry. Educators have a responsibility to the student population to keep up with the changing biochemistry laboratory. Several laboratory manuals have recently been published that reflect the myriad areas of biochemistry that can be incorporated into the undergraduate experience [1, 2]. In fact, it seems impossible to explore all the areas of biochemistry in any depth in a single-semester laboratory course. Over 400 institutions nationwide have instituted undergraduate biochemistry majors to more fully cover the range of biochemical topics [3]. In order to provide students with an exposure to both protein purification techniques and molecular biological techniques, Georgia Southern University presently offers two semesters of biochemistry within the chemistry department. Each course has a laboratory component that allows the undergraduate exposure to a wide variety of biochemical techniques. At Georgia Southern University, students gain experiences in protein purification during Biochemistry I [4] and are then led through a set of standard molecular biological techniques during Biochemistry II.

The following 3-week laboratory was developed for Georgia Southern University's Biochemistry II course. It involves the isolation of DNA from a human dried blood (DBS)11 sample, the amplification of an intron repeat sequence, and the visualization of the results using DNA gel electrophoresis to determine whether an individual is homozygous or heterozygous for the intron sequence. Finally, the students analyze the genetic and allelic frequency of the Alu repeat in the class population and compare their statistics to a U.S. sample statistic using simple Hardy-Weinberg population statistics. This procedure uniquely combines commercially available kits marketed by Bio-Rad Laboratories (Hercules, CA) to explore genetic and allelic frequencies within a population.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS AND DISCUSSION
  5. SAFETY ISSUES
  6. RELEVANT WEBSITES
  7. REFERENCES

Preliminary—

Students are informed early in the semester that they will be asked to obtain a DBS for DNA extraction. It should be noted that the use of blood samples from human subjects requires approval by the Institutional Review Board (IRB). In accordance with protocols for using human subjects in research, students are provided with an informed consent document outlining the risks and procedures that will be followed in obtaining a DBS. Procedures include the oversight of a trained health professional from the Nursing Department to assist the students in obtaining a blood sample using a standard fingerstick lancet device. One or two drops of blood are collected on a piece of filter paper. This procedure is performed during a previous laboratory period. All bodily fluid waste is considered a biohazard and must be appropriately disposed of under the guidelines of the Institutional Biohazards Committee. The simplest way of doing this is to have the enlisted health care professional collect all biohazard waste and take it with them after assisting in the blood collection. Students not wishing to participate in the blood collection are in no way penalized and are provided with a previously collected sample to use for the experiment.

Also, prior to performing the laboratory, the following topics are covered in lecture: methodology for DNA extraction, PCR assay including appropriate primer selection, DNA gel electrophoresis, and DNA fingerprinting. This information is additionally covered in the introductory material for the laboratory found in the student's laboratory manual.

Three 3-hour laboratory periods are divided as follows: Period 1: DNA extraction from a DBS followed by PCR; Period 2: DNA agarose gel electrophoresis of the PCR samples followed by staining and photographing; and Period 3: Photo data exchange among the class, data analysis of bands, and population statistical analysis.

Period 1—

The DNA extraction from a DBS is straightforward and incorporates the use of the Instagene Dry Blood Kit (Bio-Rad Laboratories; catalog no. 7326212). This kit allows the isolation of DNA from an area of dried blood the size of a ¼-inch paper hole punch from a piece of filter paper. The extraction procedure involves a straightforward, repetitive series of washing and extraction steps that remove everything from the blood on the filter paper except the DNA. This is precipitated onto the filter paper during a final ethanol treatment step.12

The primers and controls for amplification of the selected intron sequence are available commercially from Bio-Rad Laboratories as part of the Chromosome 8 PCR Biotechnology Explorer kit (catalog no. 166–0009-EDU). As background, short repetitive interspersed elements (SINES) [5] have been found in many intron sequences and are thought to have been randomly inserted into chromosomes over evolutionary time. Human chromosome 8 contains the gene for tissue plasminogen activator (PMA). The PMA gene contains one such SINE sequence located in intron 8 [57]. This repeat sequence is referred to as the Alu repeat because it contains a restriction site for AluI. The intron 8 Alu repeat in PMA is dimorphic among individuals and can thus be used to estimate insertion frequency among a population to illustrate molecular genetic variation. That is, individuals in a population can be homozygous (+/+), expressing the Alu repeat in intron 8 on both gene copies (960 bp); heterozygous (+/−), expressing the Alu repeat on just one gene copy (960 bp and 660 bp); or homozygous (−/−), meaning the Alu repeat is absent in the individual (i.e. intron 8 without the repeat = 660 bp).

After the DNA is isolated from the DBS, students must use the dilution equation to calculate how they will prepare a PCR reaction mixture containing their DBS DNA on the filter paper, the PCR reagents, and buffer from the given concentrations of the stock solutions. They are told that the final concentrations for their reaction mixture are to be: 100 mM KCl, 20 mM Tris-HCl, 4 mM MgCl2, 2 mM NTP (each), 1 μM of each primer, 2.5 units of Taq polymerase, for a total volume of 40 μl. The class prepares PCR for their samples and a set of four controls (provided in the kit). The four controls include DNA from individuals known to be (+/+), (+/−), (−/−), and a negative control containing no DNA (Neg.). The PCR mixture and the filter paper containing the DBS DNA are placed in a PCR tube, covered with a drop of mineral oil, placed in a thermocycler (MJ Research, Waltham, MA) and run through the following program: 92 °C 1 min.; 94 °C, 1 min.; 64 °C, 1 min.; 72 °C, 2 min.; 40 cycles; 72 °C,10 min. The samples and controls are kept frozen until the next laboratory period.

Period 2—

A simple, durable DNA mini-gel apparatus is available from Fisher Scientific (Pittsburgh, PA; catalog no. FB-SB-710) and works well for the DNA electrophoresis. The students prepare a 1% agarose gel in 1 × tris/acetic-acid/EDTA buffer. A 10-lane gel will accommodate two students with two PCR samples. A suggested scheme for loading the gel is shown in Table I. Forty microliters of sterile water are added to each of the four control reactions, providing enough of each control DNA for four gels (eight students). The gel is run at 100V for ∼1 h. The gel is then stained with SYBR Gold DNA Stain (Molecular Probes, Eugene, OR; catalog no. S-11494). This stain was selected because it is considered less mutagenic and more sensitive than ethidium bromide and more sensitive than methlyene blue [8]. Gels are stained for either 30 min. with agitation, or left overnight. A Polaroid (Waltham, MA) photograph is taken of the stained gels for further analysis.

Period 3—

Upon visual inspection of the gel photograph, students are able to determine whether they are (+/+), (−/−), or (+/−). The students begin their analysis of the gel by establishing a plot of the log of base pairs versus migration distance of the PCR markers. The number of base pairs corresponding to each sample band is then determined.

As previously mentioned, the Alu repeat represents a dimorphic allele that can be used to study genetic frequencies in localized populations [6]. The Hardy-Weinberg equilibrium equation, p2 + 2pq + q2 = 1, describes allelic frequency in a population, where p2 is the frequency of individuals homozygous (+/+) for the Alu insert; q2 is the frequency of homozygous individuals (−/−) lacking the Alu insert; and 2pq is the frequency of individuals heterozygous (+/−) for the Alu insert.

As an exercise in population statistics, the students determine the observed genotypic frequencies of (+/+), (+/−), and (−/−) in the class population. They then determine the allelic frequencies for their class. Students are then given the genotypic frequencies for 10,000 U.S. individuals [9] and are asked to compare the allelic frequencies for the U.S. data to the class population.

Students are required to keep notes of all laboratory procedures in a laboratory notebook, which is checked weekly for updates. Approximately 1 week after completion of the laboratory, the students are also required to turn in a technical laboratory report detailing the experiment and their results.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS AND DISCUSSION
  5. SAFETY ISSUES
  6. RELEVANT WEBSITES
  7. REFERENCES

The results of a typical student gel are shown in Fig. 1. It is clear from visual inspection of the gel that student 1 is (−/−) for the repeat (lanes 7–9) and student 2 is (+/−) (lane 10). A sample student analysis of genotypic and allelic frequency using Hardy-Weinberg statistics is shown in Table II (taken from the Spring 2003 Biochemistry II class). This analysis indicates a general agreement with the overall U.S. population sample. The students found this very satisfying because it validated their experimental results.

Prelaboratory lecture and class discussions on the subject of DNA fingerprinting work well to raise student enthusiasm for this laboratory project. Overall, this laboratory has been very successful and popular among students. However, the following pitfalls should be noted:

  • It is important that the blood collected on the filter paper be completely dried, otherwise some of the blood DNA may be removed during the initial wash step of the DNA extraction;

  • It is wise to have the students extract DNA from two dried blood samples in the event that one of their samples becomes contaminated during any one of a number of steps during the extraction procedure;

  • The straightforward, repetitive procedures of the DNA extraction are easy to follow, however, it requires some concentration to keep track of the number of times a step is repeated;

  • Loading a DNA agarose gel can be tricky for students attempting this for the first time. It is desirable that students have at least one previous experience in prepping and loading an agarose gel prior to this experiment;

  • Students must be very familiar with good, sterile pipetting techniques in order to limit the amount of material wasted during preparation of the PCR samples;

  • Obtaining meaningful population statistics may become difficult depending on the number of results and class size. However, students can still understand the principles behind the population statistics, and if not enough results are obtained by the class, a mock population statistics exercise can be performed during period 3.

Overall, this laboratory has been a positive experience for the students and has even had a positive effect on class enrollment. The Spring 2003 class has more than doubled (13 students) compared with the Spring 2002 section (six students).

This laboratory procedure represents a time-effective means for the undergraduate instructor who is not conducting research in the area of molecular biology to provide biochemistry students exposure to molecular biological techniques, using prepared kits that are relatively inexpensive and straight-forward in their set up.13 ,24

SAFETY ISSUES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS AND DISCUSSION
  5. SAFETY ISSUES
  6. RELEVANT WEBSITES
  7. REFERENCES

Students should be instructed to wear gloves when handling the dried blood samples. Any dried blood excess should be disposed in a biohazard waste container that is autoclaved prior to removal. Students should be instructed to wear gloves when handling their gel. The SYBR Gold DNA stain should be treated with caution because its toxicity is not well established [8]. As with any molecular biology procedure, appropriate sterile techniques should be followed. A listing of these procedures can be found on page 69 of the instruction manual for the Biotechnology Explorer Kit [9].

RELEVANT WEBSITES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS AND DISCUSSION
  5. SAFETY ISSUES
  6. RELEVANT WEBSITES
  7. REFERENCES

Bio-Rad Laboratories (company website) (2003) Life Biotechnology Explorer Kits (Life Science Education/Classroom Kits/Chromosome 8 PCR Kit Curriculum): www.bio-rad.com

Bio-Rad Laboratories (company website) (2003) LifePV92 PCR Informatics Kit Curriculum (Life Science Education/Free Biotechnology Explorer Curricula/PV92 PCR Informatics Kit): www.bio-rad.com.

Bio-Rad Laboratories (company website) (2003) Instagene Dry Blood Kit (Clinical Diagnostics/Genetic Disorders Testing/DNA Diagnostic Kits and Reagents/DNA Sample Preparation Reagents/Instagene DNA Extraction Kits): www.bio-rad.com

thumbnail image

Figure FIGURE 1.. Products of PCR amplification of the Alu-PMA intron 8 from chromosome 8.Lane 1 contains marker DNA, lanes 3–6 contain controls, lanes 7–10 contain student samples. Sample 1 is homozygous (−/−) and sample 2 is heterozygous (+/−) for the Alu repeat. The band at ∼1700 bp in sample 2a represents heteroduplex DNA.

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Table Table I. Suggested DNA gel electrophoresis lanes for a 10-lane gel
Lane12345678910
SampleMarkers+/+−/−+/−Neg.DBS1aDBS1bMarkersDBS2aDBS2b
Volume (ul)10202020202020102020
Table Table II. Observed 2003 spring class genotypic and allelic frequencies compared with sample U.S. data [9]
GenotypeVariable in Hardy-WeinbergNo. class genotypesaClass genotypic frequencybNo. U.S. genotypesU.S. genotypic frequency
(+/+)p24.36362,422.2422
(−/−)q23.27282,050.2050
(+/−)2pq4.36365,528.5528
AlleleEquationNo. class allelesClass allelic frequencycNo. U.S. allelesU.S. allelic frequency
  • a

    a From a class of 13 students, two students could not determine whether they were heterozygous or homozygous from the results of their gel.

  • b

    b Genotype/total.

  • c

    c Allele type/total.

+ = pP = 2(p2) + 2pq12.544510,372.5186
− = qq = 2(q2) + 2pq10.45459,628.4814
  • 1

    The abbreviations used are: DBS, dried blood sample; IRB, Institutional Review Board; SINE, short repetitive interspersed elements; PMA, plasminogen activator.

  • 2

    As an alternative to extracting the DNA from a dried blood sample, instructors may wish to use DNA from cheek cells. This procedure is provided in the Bio-Rad Biotechnology Explorer Kit (www.bio-rad.com/Life Science Education/Free Biotechnology Explorer Curricula/PV92 PCR Informatics Kit). The use of cheek cells does not require IRB approval. The motivation for using the dried blood was to capture student interest, demonstrate how robust DNA can be, and show that even with a small amount of an older DNA sample, DNA can be effectively extracted.

  • 3

    All relevant set-up procedures are outlined in the Protocols for the Dried Blood Kit and the Biotechnology Kit Curriculum (see Relevant Websites).

  • 4

    Since the development of this laboratory, Bio-Rad Laboratories has begun marketing a second set of primers and controls to amplify a different Alu-repeat found in PV92 on chromosome 16 (catalog no. 166–2100-EDU) [10]. The laboratory portion of this kit follows the same protocol as the Alu-PMA repeat on chromosome 8 described here.

REFERENCES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS AND DISCUSSION
  5. SAFETY ISSUES
  6. RELEVANT WEBSITES
  7. REFERENCES
  • 1
    A. J. Ninfa, D. P. Ballou (1998) Fundamental Laboratory Approaches for Biochemistry and Biotechnology, Fitzgerald Science Press, Bethesda, MD.
  • 2
    C. Hardin, J. Pinczes, A. Riell, D. Presutti, W. Miller, D. Robertson (2001) Cloning, Gene Expression and Protein Purification: Experimental Procedures and Process Rationale, Oxford University Press, New York.
  • 3
    American Society for Biochemistry and Molecular Biology (2003) List of Schools: Departments with Degree Programs in Biochemistry and/or Molecular Biology: www.asbmb.org.
  • 4
    M. O. Hurst, M. V. Keenan, C. C. Son (1992) Purification of lysozyme by linear salt gradient and SDS gel electrophoresis, J. Chem. Ed. 69, 850851.
  • 5
    P. L. Deininger, in M.Howe, D.Berg, Eds. (1989) Mobile DNA, ASM Press, Washington, D.C., pp. 619636.
  • 6
    S. A. Tishkoff, G. Ruano, J. R. Kidd, K. K. Kidd (1996) Distribution and frequency of a polymorphic Alu insertion at the plasminogen activator locus in humans, Hum. Genet. 97, 759764.
  • 7
    S. J. Degen, B. Fajput, E. Reich (1986) The human tissue plasminogen activaor gene, J. Biol. Chem. 261, 69726985.
  • 8
    Molecular Probes (company website) (2003) Product information sheet, SYBR Nucleic Acid Gel Stains: www.probes.com/media/pis/td004.pdf.
  • 9
    Bio-Rad Laboratories (company website) (2003) Chromosome 8: Gene Amplification Alu-TPA PCR Kit Chromosome 8 PCR Kit (Catalogs/Life Science Education/Classroom Kits/Chromosome 8 PCR Kit): www.bio-rad.com.
  • 10
    Bio-Rad Laboratories (company website) (2003) Chromosome 16: PV92 PCR/Informatics Kit (Catalogs/Life Science Education/US Workshops and Conferences): www.bio-rad.com.