mtDNA Amplification and Comparison
Overview and concepts
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, r
un 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
DNA extraction, PCR, DNA sequencing, mutations, mutation rates, phylogenetics
Prior knowledge required
Types of mutations
point, frameshift, etc
tion ~ 20 min
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
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
Go PCR Beads, DI water, forward primer, reverse primer, DNA sample, microcentrifuge,
thermocycler, vortex machine (not essential)
DNA Extraction. Use DNA Extraction worksheet (see attached documents).
PCR . Use the PCR Procedure
worksheet (see attached documents)
Send samples to ColdSpring for sequencing.
Creating Primers. Use worksheet (see attached documents).
Do Primer3 activity before
handing out PCR procedure worksheet, as the PCR worksheet contains the
sequence for the primers used.
mtDNA Comparison: Will be ~ 1 week after PCR procedure to give time for DNA to be sent
off and sequences returned.
Use the mtDNA Comparisons worksheet (see attached documents
Use the website to obtain student
Use Dolan site for phylogenetic activity.
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.
You can use Science in Motion to borrow necessary equipment if you do not have it readily
available in your school.
You can order materials for the procedure (Carolina and WARDs each sell kits/materials) or you
can make your own.
Discussions, quizzes and exams, progress of and final work on the lesson/assignment
Have students compare their mtDNA sequences to the Cambridge Reference Sequence and note
) provides a great animation of the
process of PCR
Revised Cambridge Reference Sequence
Contains the revised Cambridge Reference Sequence (rCRS) of Human mtDNA.
Primer Design Guidelines
) Gives a guideline for
how to create/choose good primers for optimum results.
Dolan Learning Center’s S
equence Server Database
DNA Sequencing Services
Click on the link on the menu at
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.