Is Is There a Ge Genetic Ba Basi sis t s to R Race? Exam amining human an var ariation an and an ancestry usi using ng mt mtDNA CCSF Mission Campus February 10, 2018
No Note t to t teache achers: : the following slides are background info for teachers as given during a BABEC workshop. Student sections start on slide #9.
Objectives (for teac achers) 1. Beta-test of new template-go over lesson packet 2. New approach that addresses NGSS 3-Dimensions 3. New curricular concepts – why? a. Popularity of personal genomics/commercial ancestry tests b. Uses scientific evidence to dispel societal misconceptions c. Uses the potentially charged topic of race to connect and engage students with modern society and current methods in genetics d. Idea : Genetic variation within any race is greater than between races We want YOUR feedback!
Ho How we e de devel eloped ped thi his les esson Genetic Origins curriculum from DNA Learning Power of Illusion PBS Documentary New updated version with approachable bioinformatics
BABEC’s ad adap aptation • Prompts students to examine their assumptions about the link between genetics and race • Provides an opportunity for students to examine their own deep ancestry by discovering their haplogroup • Examines whether students’ own mtDNA is more/less similar to their peers • Analyzes similarity in DNA sequences using a bioinformatics platform widely used by the scientific community (CLUSL Omega) • Follows 3-Dimensions of NGSS
Thr Three ee Dimens ensions ns of NGSS HS-LS3-2 Make and defend a claim based on evidence that heritable genetic variation may result from: (1) new genetic combinations through meiosis, (2) viable errors occurring during replication, and/or (3) mutations by environmental factors. SEP: Engaging in Argument from Evidence DCI: LS3.B Variation of Traits Cross-Cutting Concept: Cause and Effect Evidence Statement from HS-LS3-2
Le Let’s Star art! t!
Is Is There a Ge Genetic Ba Basi sis t s to R Race? Exam amining human an var ariation an and an ancestry usi using ng mt mtDNA
Read th the challenge statement t below: Th There i is a s a g genetic b basi sis t s to r race . Do you agree or disagree and why? Ø Write your answer on your lab notebook. Ø Discuss…
Video: Race, the Power of Illusion, Episode 1 Newsreel (0:45-4:37) https://www.youtube.com/watch?v=Y8MS6zubIaQ&t=3s Ø Watch Video Ø Fill out the worksheet, answering… Ø Who in this room are you most biologically similar to? Ø Who are you least biologically similar to? Ø Why do you think so? Discuss… Ø Next step, Lab activity!
Lab ab Activity Workflow 1) Extract your cheek cell DNA using Chelex 2) Amplify 440-nucleotide sequence from the D-loop 4) Analyze your mtDNA sequence of your mt genome using bioinformatics 3) Submit PCR samples for sequencing at CSUEB
PCR is DNA Replication in a a Test Tube! Polymerase Chain Cellular DNA Reaction Replication What are we copying? DNA DNA Heat Enzymes How do we separate the DNA? What is doing the copying? Taq polymerase Human polymerase How do we fish out the sequence? Primers Primers What does the work? Thermal cycler Cell
Why Wh y do we do PCR? Individual genes are present in amounts too After 30 cycles, Cycle 3 low to be detected in vivo . DNA is amplified over a billion fold. PCR amplification allows for their detection and measurement from a very small sample It produces the double amount of product Cycle 2 from the previous cycle, for an exponential increase Cycle 1
DNA DNA Sequencin cing – its eas asy!
San anger Sequencing Animations From DNALC https://www.dnalc.org/resources/animations/sangerseq.html DNA Sequencing in 3D https://www.youtube.com/watch?v=ONGdehkB8jU The Di-deoxy Approach https://www.youtube.com/watch?v=bEFLBf5WEtc
Sequence Data a Results & Bioinformatics o You will receive sequence files from CSUEB with the extension “.ab1” o These files need special software to open SnapGene is open source Sequence chromatogram Sequence text file è è ! Once you get your text file, you can do all kinds of fun stuff!
But first – the lab activity!
Lab Flowchart: Chelex DNA Isolation Procedure Rinse mouth Transfer saline Pour off liquid Resuspend Centrifuge with saline Mix Boil Centrifuge Transfer cells into Chelex Transfer DNA out of Chelex Store on ice Fall 2011, page 1
Introduction to microfuge tubes A microfuge tube 1500 µ l has a total volume of 1.5 milliliters (ml), or 1000 µ l 1500 microliters ( µ l). 500 µ l 100 µ l 1000 µ l 1500 µ l Fill the tube with your saline rinse to a volume of 1000 µ l to 1500 µ l (step 4) Fall 2011, page 2
Ideal Centrifuging Method Orient the hinge of the tube to point outward and away from the middle of the centrifuge. This allows for solid material to settle to the bottom of the tube directly below the hinge. Hinge of tube points out All spins should be performed at 10,000 x g, which is about 10,000 rpm depending on the centrifuge If you have a less powerful centrifuge, spin longer than 5 minutes After centrifuging, look below the hinge for the solid material (pellet) Fall 2011, page 3
The Cell Pellet After you centrifuge the saline rinse, your cheek cells will settle to the bottom of the tube. This is called a cell pellet . The cell pellet will be white. Observe your cell pellet. If you do not see one, add more saline rinse and repeat. The clear liquid above the cell pellet is called the supernatant . It does not contain any cells. When you place your tube in the centrifuge, have the hinge of the tube facing outward. Then you can find your cell pellet directly underneath the hinge. Cell pellet back view Cell pellet side view Supernatant Cell Pellet Fall 2011, page 4
Decanting the Supernatant The supernatant can be poured out of the tube by inverting it. The cell pellet will stick to the bottom of the tube while you pour. (step 6) Observe cell pellet stuck to the bottom of the tube while you pour. If you do not see one, repeat with more saline rinse. Fall 2011, page 5
Resuspending the Cell Pellet To resuspend means to dislodge the cell pellet and mix in into the liquid using your pipette. There will be about 100 µ l of supernatant remaining in the tube after you pour. 100 µ l Look at the 0.1 line on the bottom of the tube and add more saline if it is not at that level. (step 7) After resuspension, the solution will be cloudy. This is because you have concentrated your cells into a smaller volume than what you started with. Fall 2011, page 6
Chelex Chelex is made of very small white beads. They are heavy and settle to the bottom on the tube. Chelex can � t be pipetted with a P20 or P200 tip. It will shear the beads. Use a P1000. Tiny Chelex beads Chelex beads mixed with your cell suspension (step 9) Fall 2011, page 7
Removing the DNA After heating and centrifuging, DNA is in the supernatant. DNA is in the supernatant Chelex beads & cell debris Remove DNA with a pipette Keep pipette tip near the top (step 13) of the liquid while aspirating Keep the pipette tip near the surface of the liquid so as to not touch the Chelex beads. Fall 2011, page 8
PCR Reaction Set-up ü 20 µl of Master Mix Contains buffer, dNTPs, MgCl 2 , AmpliTaq Gold ü 20 µl of Primer Mix Contains the forward and reverse primers, diluted to the proper concentration ü 10 µl of your own DNA
Experimental controls For every experiment, a positive and negative control should be done in order to produce credible data. ü Positive control: human DNA of known genotype o Will produce a result that shows all possible positive results in an experiment o Will show that the reagents are working as expected ü Negative control : water in place of DNA o This reaction lets us know if we have any contaminated reagents
Start on page 4 of the new curricula, and we would love your feedback!
Knowledge an and bioinformatics section Ø Background Lecture Ø Bioinformatics Activities ü Haplogroups using Mitomap ü Sequence comparison using Clustal Omega Ø Electrophoresis Results
Mitochondria, a, our “second” genome Food + O 2 = Energy + CO 2 + H 2 O (glucose) (ATP) Powerhouse of the Cell Images: DNLC & Genebase
mt mtDNA Co Control R Region / / D D-Loop p / Hype ypervariabl ble Regi gion A gene-free region of ≈1,000 nts like a promoter or origin of replication accumulates point mutations at � 10x rate of nuclear DNA It is our fastest evolving DNA sequence! Over evolutionary time, many more mutations have accumulated in the D-Loop than the coding region. Why? Image: Genebase
mt mtDNA is Maternal ally In Inherited Why is this significant for ancestral studies? 1) It is not subject to recombination: stays the same throughout generations 2) Changes only occur through mutation, which is passed on 3) Has a strict line of descent from mother to child Image: Genebase
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