Tracing Your Matrilineal Ancestry: Mitochondrial DNA PCR and - - PowerPoint PPT Presentation

Tracing Your Matrilineal Ancestry: Mitochondrial DNA PCR and Sequencing

BABEC’s Curriculum

• Rewrite Curriculum to Align with NGSS

Standards

• NGSS work group
• Your ideas for incorporating NGSS
• Feedback from you (evaluation at end
• f workshop)

Using a Storyline

NGSS Standards

• HS-LS4-1. Communicate scientific information

that common ancestry and biological evolution are supported by multiple lines of empirical evidence.

• HS-LS3-1. Ask questions to clarify relationships

about the role of DNA and chromosomes in coding the instructions for characteristic traits passed from parents to offspring.

…more after lunch

Pipetting Skills P1000 P200 P20

Lab Workflow

1) Extract your cheek cell DNA using Chelex 2) Amplify 440-nucleotide sequence from the D-loop

• f your mt genome

3) Submit PCR samples for sequencing at CSUEB 4) Analyze your mtDNA sequence

Lab Activity

Follow with your protocol…

How do we start this storyline?

• Ask questions. Who are you? Where do you

come from? Where are your ancestors from? How do you find out?

• Use video?

https://genographic.nationalgeographic.com/f

• r-educators/ “About the Project” first 25

seconds

• Any ideas?

What is the Polymerase Chain Reaction

We use this method to copy DNA in vitro to detect specific genes of interest Why? Individual genes are present in amounts too low to be detected in vivo. PCR amplification allows for their detection and measurement from a small sample It produces the double amount of product from the previous cycle, for an exponential increase

A technique in molecular biology used to rapidly amplify a piece of DNA across several orders of magnitude, generating millions of copies of a specific DNA sequence

Image: Aim Shams Univ http://www.obgynacademy.com/basicsciences/fetology/genetics/

Exponential Amplification of Target DNA Sequences

Cycle 1 Cycle 2 Cycle 3 After 30 cycles, DNA is amplified

• ver a billion fold.

PCR products are called amplicons

PCR Reaction Components

All components are needed in optimal concentrations Animation: 1.http://passel.unl.edu/pages/informationmodule.php?idinformationmodule =968252315&topicorder=3&maxto=11

• 2. https://www.dnalc.org/resources/animations/

PCR vs Cellular DNA Replication

Polymerase Chain Reaction Cell What are we copying? How do we separate the DNA? What is doing the copying? How do we fish out the sequence? What does the work? Cellular DNA Replication DNA DNA Heat Enzymes Taq polymerase Human polymerase Primers Primers Thermal cycler Cell

DNA Sequencing

Images: UCI, Genebase, ABI

Sanger Sequencing Animation

From DNALC

• https://www.dnalc.org/resources/animations/

sangerseq.html

Sequence Data Results

You will receive sequence files from CSUEB with the extension “.ab1” They need special software to open è its open source and free!

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Sequence chromatogram (.ab1) Sequence text file (.txt)

The Science behind this Module

Mitochondria, our “second” genome

Food + O2 = Energy + CO2 + H2O (glucose) (ATP)

Images: DNLC & Genebase

Powerhouse of the Cell

mtDNA Control Region / D-Loop / Hypervariable Region

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

mtDNA is Maternally 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

Unique Properties of mtDNA

We have many more copies of mtDNA than other DNA Large amount of mtDNA & its small size = excellent candidate for anthropological studies of old or degraded samples Maternal inheritance gives us specific information about human migration & evolution Because they don't mix with genes from the father's line, mt genes can be used to trace a sort of lineage right the way back to when our first ancestors came out of Africa. The region we sequence has a high mutation rate. It is our fastest evolving DNA sequence!

Deep Ancestry

Common Maternal Ancestors people with the same mtDNA SNPs as you

Images: Wikipedia, BBC

Ancestral Marker a mutation that occurred a long time ago Single Nucleotide Polymorphism

• ne nucleotide replaced by another

SNPs are the mutations found in mtDNA

Deep Ancestry: ancestry from tens of thousands of years ago

Human Origins & Migration

≈27 major groups compromise the human race

Haplogroup Groups or people with similar haplotypes - they share a common ancestor Haplotype inheritance of a cluster of single nucleotide polymorphisms (SNPs)

https://genographic.nationalgeographic.com/science-behind/ The Journey of Your Past Video

Migration Map Interactive

• https://genographic.nationalgeographic.com/

human-journey/

Identifying Your SNPs

The position of any base pair in the mtDNA is designated by counting from “1ʺ clockwise around the mtDNA Nucleotides in HVR1 region are in locations 16001 to 16520 (low resolution)

Bioinformatics Workflow

1. Obtain .ab1 file 2. Crop off the bad reads using SnapViewer 3. Save as text file (.txt file) 4. BLAST against rCRS; write down SNPs 5. Use mtDNA Haplotype finder at: www.mitomap.org/foswiki/bin/view/MITOMASTER/WebHome 6. Find your Haplotype!

Bad Reads

mtDNA Sequence References

• Human mtDNA first sequenced in 1981 (called

the Cambridge Reference Sequence - CRS)

• Revised sequence in 1999 (revised CRS – rCRS)
• Differences are explained here:

http://www.mitomap.org/MITOMAP/Human MitoSeq

• 2012 – a Reconstructed Sapiens Reference

Sequence (RSRS)

– Uses “mitochondrial Eve” as root

Phylotree.org http://haplogrep.uibk.ac.at/blog/rcrs-vs-rsrs-vs-hg19/

revised Cambridge Reference Sequence (rCRS)

The first human mtDNA fully sequenced; 1981 Your SNPs are determined based on comparison with CRS your sequence rCRS 3 SNPs: positions 16278(CèT), 16311(TèC), & 16362(TèC)