Human Single Nucleotide Polymorphism Determination
through its collaboration with the SERC Pedagogic Service.
In this laboratory exercise, students determine which allelic form of a particular single nucleotide polymorphism (SNP) they have (one located in an intron, and not associated with any known phenotype). Students may be homozygous for the A/T pair or the G/C pair, or they may be heterozygous with A/T on one chromosome and G/C on the homologous chromosome. Students isolate their own DNA, perform polymerase chain reaction (PCR) to amplify a region surrounding this SNP, and use RFLP (restriction fragment length polymorphism) analysis to determine their genotype.
- Students will understand the importance of SNPs as medical diagnostic indicators and their applicability to studies of human evolution.
- Students will gain an understanding of using PCR to amplify a specific segment of DNA.
- Students will enhance their understanding of DNA replication by learning about the technique of PCR.
- Students will use RFLP analysis to detect different forms of a SNP in their own DNA.
Context for Use
This activity is a part of an introductory-level undergraduate biology course on genes, evolution, and development. Students should understand basic DNA structure and base pairing. The activity is used to help students understand PCR and SNPs. It could easily be modified to introduce restriction enzymes and gel electrophoresis (see Teaching Notes and Tips below), though as written we use it to reinforce students' prior understanding of these tools. This lab would also be appropriate to a mid-level genetics course.
This lab is designed to support a SNP case study ( This site may be offline. ) which is an important part of our course. The lab stands alone nicely, but can also be used with the case study to firmly connect lab and course work together with a thoughtful discussion of ethical implications of human SNP analysis. Students are excited by the relevance both these activities have to their lives.
Description and Teaching Materials
Single nucleotide polymorphisms (SNPs) in the human genome have the potential to be powerful diagnostic indicators in human health, for predicting both disease susceptibility and drug effectiveness. SNPs are also being used in studies of human migration and evolution. This activity investigating one particular human SNP is broken up into two lab sessions as described below.
First lab session:
- Students isolate their own cheek cell DNA using a kit (60 minutes).
- Students amplify a particular 311 base pair segment of DNA which contains one SNP (an A/T pair on the chromosomes of some people and a G/C pair in other individuals). Students set up a PCR reaction using ready-made master mix in beads (15 minutes). The PCR reaction runs in a thermal cycler after the students leave the lab, and an instructor moves the tubes to the freezer until the next lab session.
Second lab session:
- Students perform a restriction enzyme digest of their PCR product (100 minutes, including a 90 minute incubation). The restriction enzyme leaves the A/T variant of the SNP intact but cuts the C/G variant, leaving fragments of 94 and 217 base pairs.
- Students run both their cut and uncut product on a gel along with a known control (90 minutes, including 55 minutes to run the gel).
Student Lab Handout (Word) (Microsoft Word 58kB Apr17 07)Student Lab Handout (pdf) (Acrobat (PDF) 496kB Apr25 07) This is the lab handout given to the students, which explains the DNA isolation, PCR, digest, and gel electrophoresis procedures in detail. It includes background information for the students, as well as space for them to work out the PCR primers used and the RFLP results they would expect for an A/T homozygote, a C/G homozygote, and a heterozygote.
Teaching Notes and Tips
Integrating the Lab Experience
This lab is integrated into our course in several ways. One of the most fundamental ways that information about SNPs is reinforced during the course is with the SNP case study ( This site may be offline. ) , in which students role play positions on a bill concerning genetic privacy. This lab activity ties into the course in numerous smaller ways as well, including the location of this SNP, the relationship between PCR and DNA replication, links between the fields of genetics and evolution, and a focus on human biology.
- SNP selection. The SNP we chose to focus on in the lab is found in the intron region of a cell cycle protein we discuss in class.
- PCR and DNA replication. We have found that teaching the concept of PCR after we have introduced DNA replication, and asking students to compare and contrast the processes, cements their understanding of both: they start thinking less abstractly about what elements are required for DNA to be copied.
- Genetics and evolution.Throughout our particular course, we connect the fields of genetics and evolution. We strengthen that connection by pointing out that SNPs are used to study human evolutionary history.
- Human biology. We often use examples from human biology in our course, and the SNP lab gives us a chance to put that into practice. Students find it intrinsically interesting that SNPs can be used to help in the field of medicine. While we encourage this interest, we have been very careful in the lab activity to select a particular SNP which has no known link to disease, drug susceptibility, or any other trait. We want this lab to focus the students on the process of SNP determination, and to help them understand the possibilities SNP data contain, but we do not want to expose the students to information about themselves (or their peers) which would be better discussed with a genetic counselor.
We chose this SNP by searching the SNPper web site, which allows multiple types of searches for SNPs, including by gene name. We selected a SNP using three main criteria:
- The SNP was in a gene coding for a cell cycle protein we talked about in class (CDK3).
- The SNP was in an intron region with no known diagnostic value.
- The SNP had known allele frequencies in multiple populations, and the two alleles were both common. (We did not want a SNP with few heterozygotes, or with one allele only present in some identifiable subset of the students in our course.)
SNPs are being extensively analyzed as markers for disease susceptibility. Particular SNPs have been associated with diseases, Crohn's Disease being one recent example. In addition, broader patterns of SNPs (haplotypes) are being analyzed as possible indicators of disease susceptibility. The International HapMap Project is collecting data from multiple populations for this purpose. They published "A haplotype map of the human genome" in Nature in 2005. A good description of their efforts and potential applications can be found in the accompanying summary "Genomics: Understanding human diversity".
In addition to supporting medical research, SNP analysis can be used to explore human evolution and early migration. The Genographic Project is collecting SNP information from multiple populations around the world (as well as volunteers, who can pay to get a DNA collection kit and participate). This information is being used to map early routes of human migration.
Information for Lab Instructors and TAs
TA and Instructor Notes (Word) (Microsoft Word 57kB Apr24 07) TA and Instructor Notes (pdf) (Acrobat (PDF) 215kB Apr24 07) This provides a detailed description of how to run the lab, including information about the PCR primers and expected fragment sizes.
We have provided an example gel showing the marker lane and digested and undigested heterozygote DNA.
Equipment, Supplies, and Lab Set-Up
A detailed list of the set-up necessary to perform the lab, including equipment, supplies, and sources of material.
Modifications to the Lab
Here are additional resources which could easily be added into the lab:
- Additional SNP background information for students, including basic information about what SNPs are and how they are being used in biological and medical research.
- Restriction Enzymes (Word) (Microsoft Word 29kB Apr24 07) Restriction Enzymes (pdf) (Acrobat (PDF) 85kB Apr24 07) This file contains the background information we provide to our students about restriction enzymes earlier in our course.
- Gel Electrophoresis (Word) (Microsoft Word 81kB Apr24 07) Gel Electrophoresis (pdf) (Acrobat (PDF) 333kB Apr24 07) This file contains the background information we provide to our students about gel electrophoresis earlier in our course.
Student understanding of these concepts and procedures is assessed in two main formats:
- Students complete study questions as part of the SNP case study and turn these in as a homework assignment. One of the study questions asks directly about RFLP analysis of SNPs. The questions and an answer key are available along with the case study ( This site may be offline. ) .
- The students' understanding of PCR is tested in a course exam and also in an ungraded assessment completed at the end of the term.
References and Resources
"SNPs and Snails and Puppy Dog's Tails, and That's What People Are Made of..." A Case Study on Genome Privacy ( This site may be offline. ) is available from the National Center for Case Study Teaching in Science (more info) .
The National Genographic Project uses SNPs to determine the earliest patterns of human migration.
The International HapMap Project is using SNPs to learn more about genetic variation in humans, and what implications this variation has for medicine.
A haplotype map of the human genome, based on information from SNPs, was published in Nature (more info) in 2005. Genomics: Understanding Human Diversity is a more accessible description of this publication.