mtDNA Amplification and Comparison
Overview and concepts
Overview
This series of lessons is designed to introduce students to PCR – its function and the process. Students
will have hands-on experience extracting their own DNA, working with an online computer program to
create primers for their targeted region of mtDNA, run a PCR reaction, and compare their own mtDNA
with ancient and modern human mtDNA to understand the mutation rate of DNA, the concept of
molecular clocks, and the relationships among various human groups and between human groups and
animal groups.
Grade level
Grades 11-12.
Concepts covered
DNA extraction, PCR, DNA sequencing, mutations, mutation rates, phylogenetics
Prior knowledge required:
DNA structure
DNA replication
Types of mutations – point, frameshift, etc
Activity notes
Time frame
 DNA extraction ~ 20 min
 PCR mini-lecture ~ 15 min (can do after setting up PCR to run)
 Creating Primers / Primer-3 ~ 30 min
 PCR ~ 10 min set-up (1 hr to run)
 mtDNA comparisons ~ 80 mi
Materials:
worksheets (DNA extraction, Creating Primers, PCR Procedure, mtDNA Comparisons), computers with
Internet access, materials for DNA extraction (saline solution, plastic cups, 1.5 ml tubes, micropipettes,
microcentrifuge, 10% Chelex solution, boiling water), materials for PCR (micropipettes, Ready-To-Go PCR
tubes, Ready-To-Go PCR Beads, DI water, forward primer, reverse primer, DNA sample, microcentrifuge,
thermocycler, vortex machine (not essential)
Teaching Tips
 Day 1 –
o
o
o

DNA Extraction. Use DNA Extraction worksheet (see attached documents).
PCR . Use the PCR Procedure worksheet (see attached documents)
Send samples to ColdSpring for sequencing.
Day 2 –
o
PCR lecture
o


Creating Primers. Use worksheet (see attached documents). Do Primer3 activity before
handing out PCR procedure worksheet, as the PCR worksheet contains the actual
sequence for the primers used.
Day 3 - mtDNA Comparison: Will be ~ 1 week after PCR procedure to give time for DNA to be sent
off and sequences returned.
o Use the mtDNA Comparisons worksheet (see attached documents
o Use the website to obtain student results.
o Use Dolan site for phylogenetic activity.
o Students will need to make sure they only enter the most reliable portion (that which
does not have a lot of discrepancy on bases) of their sequence into the BioServer when
doing mtDNA comparison.
For PCR Amplification:
o You can use Science in Motion to borrow necessary equipment if you do not have it readily
available in your school.
o You can order materials for the procedure (Carolina and WARDs each sell kits/materials) or you
can make your own.
Assessment
Discussions, quizzes and exams, progress of and final work on the lesson/assignment
Extensions
 Have students compare their mtDNA sequences to the Cambridge Reference Sequence and note
any differences.
Resources
 PCR Animation – (http://www.karymullis.com/pcr.shtml) provides a great animation of the
process of PCR
 Revised Cambridge Reference Sequence – (http://www.ncbi.nlm.nih.gov/nuccore/251831106)
Contains the revised Cambridge Reference Sequence (rCRS) of Human mtDNA.

Primer Design Guidelines (http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html) Gives a guideline for
how to create/choose good primers for optimum results.


Primer 3 - http://biotools.umassmed.edu/bioapps/primer3_www.cgi
Dolan Learning Center’s Sequence Server Database
http://www.bioservers.org/bioserver/index1.html

DNA Sequencing Services – Click on the link on the menu at
http://www.geneticorigins.org/mito/mitoframeset.htm
Acknowledgments
These teacher notes and resources were produced, modified, and collected by Rachel Hanner. Some of
the procedures were taken/modified from those used during Franklin and Marshall’s Bioinformatics
Seminar for High School Teachers.