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Introduction to Biotechnology: An Essential Curriculum, Page 3

Getting Started
DNA structure and function
Basic lab techniques
Restriction analysis
PCR and sequencing
Biotechnology vocabulary assessment
Optional labs and activities

PCR and sequencing

Polymerase chain reaction (PCR)is an essential tool of biotechnology researchers and is what made the Human Genome Project possible. It allows scientists to amplify (make billions of copies of) a DNA sample for analysis and experimentation. If you do not have the capacity to perform real PCR in your lab or classroom, then do a simulation.

The PCR Forensics Simulation Kit offers a fine PCR simulation that introduces the topic of PCR and reinforces your students' electrophoresis skills. Follow up this lab by encouraging your students to visit Cold Spring Harbor's DNA Learning Center Web site. It has an excellent animated explanation of PCR (and other DNA-related processes) that can reinforce classroom learning and help resolve any lingering questions.

If you are looking for an affordable and easy way to do real PCR, we recommend the Amplification of Lambda DNA by PCR Kit. With this kit, students do an actual, hands-on, PCR experiment without an expensive thermocycler (a PCR machine). They perform a timecourse amplification of a 1106-bp segment of the lambda genome by using 2 water baths, allowing results to be seen from cycle to cycle. This is a foolproof lab that can tolerate student errors and still produce good results. Another benefit of this lab is that it does not require expensive pipettors, so you can save money by using the Pipetting Device instead.

Advanced Technology (AT) Human DNA Typing by PCR™ Kits

All of the followin (AT) PCR™ labs utilize Ready-To-Go-Beads (RTGB), which reduce preparation time and student error. These labs require the use of a thermocycler with a heated lid such as the MultiGene Gradient Thermal Cycler. A thermocycler is a worthwhile investment considering the time and effort you and your students invest in a PCR lab. If your school cannot afford one, then we suggest you

  • borrow one from a nearby university, hospital, business, or school;
  • ask your district to purchase one machine to be shared among the schools in the district; or
  • get financial assistance from your state's education department. (Remind them that technology money can be used for instruments as well as for computers.)

Each PCR lab involves 5 basic components.

  1. Isolating DNA from human cells (hair sheaths or cheek cells).
  2. Utilizing specific primers to amplify the chosen locus by PCR.
  3. Analyzing the amplified DNA by gel electrophoresis.
  4. Analyzing and discussing the results of the lab.
  5. Extending the lab with bioinformatics (computer analysis).

A typical class schedule for a PCR lab includes the following:

  • Day 1—Prelab discussion
  • Day 2—DNA isolation
  • Day 3—PCR amplification (samples typically run overnight)
  • Day 4—Electrophoresis: load, run, stain, and photograph gels
  • Day 5—Results and discussion
  • Day 6—Extension activities on the Dolan DNA Learning Center Web site

Try to do at least one of the following 3 (AT) PCR labs:

  • The Human Mitochondrial DNA Kit (AT) should be the first one you do if you are going to do all 3. This lab utilizes mitochondrial DNA (mtDNA) from students' own cells. Because there are numerous mitochondria in each cell, there is a large quantity of DNA available for amplification, which means that this lab is more tolerant of student errors than the following labs are. It also works well with hand cycling due to the greater amounts of DNA used, and the number of cycles can be shortened if necessary.

    Students extract and amplify a 460-nucleotide sequence of DNA from within the control region of the mitochondrial genome of their own cells. These control regions have been widely used to study human evolution. As a bonus, amplified student samples may be submitted to the DNA Learning Center (DNALC) Sequencing Service, which will generate students' individual DNA sequences and post the results on the Web. There is no charge for this service. Students can then compare and analyze their sequences with those of other students and populations across the U.S. and around the world. Extension activities such as "The Neanderthal Mystery" and "The Mystery of the Romanovs" are also found on the DNALC Web site.
  • If you plan to do only one PCR lab, do this one. The Human Alu Insertion Polymorphism Kit AT allows students to visualize their own DNA and to compare their "DNA fingerprints" with those of their classmates. The lab checks for the presence (+) or absence  (-) of a ~300-bp transposable Alu element located on chromosome 16. This lab lends itself to population studies, Hardy-Weinberg distributions, and human evolution studies. Facilities for these extension studies can be found at the DNALC Web site.
  • The Human VNTR Polymorphism Kit AT also allows students to examine their own DNA. This lab checks for a variable number of tandem repeats (VNTR) polymorphism, which is caused by short, repeated copies of a 16-nucleotide sequence at the pMCT118 locus on chromosome 1. Differences in the number of repeated units produce longer or shorter alleles, which show up in the electrophoresis gel as bands of various lengths. Because there are 29 known alleles, a class of students shows a variety of different genotypes. This illustrates the use of DNA fingerprinting to identify individuals in court cases and disasters. This lab is more sensitive than the Human Alu Insertion lab, so if your students do both, be sure they do this one last so that their skills and techniques will be a little sharper.
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