Teacher Notes - Bioinformatics Activity Bank

stalliongrapevineBiotechnology

Oct 1, 2013 (3 years and 11 months ago)

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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, 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
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 extrac
tion ~ 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

DNA Extraction. Use DNA Extraction worksheet (see attached documents).

o

PCR . Use the PCR Procedure
worksheet (see attached documents)

o

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.nl
m.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 S
equence 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


Acknowl
edgments

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.