The answer is in the DNA

Neil Chitrao, Arun Chakravorty and Eric Holmes

Cornell iGEM1

Who are we?• Neil Chitrao: aka Captain America

o Sophomore biological science and French major

o Wetlab Team Member

• Arun Chakravorty: aka Dr. Watsono Junior biological science major

o Wetlab Team Leader

• Eric Holmes: aka Sherlocko Junior biological engineering major

o Full Team Leader

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Cornell iGEM Team● Award-winning

undergraduate project

team

● International Genetically

Engineered Machine

● “Biobricks” : Standardized

genetic parts

○ Ever-expanding registry

● Annual competition

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Trouble in Burkholderia• For decades, the United Nations has exhorted the autocratic

government of Burkholderia to send representatives to New York City

in response to allegations of severe human rights abuses

• In a surprising turn of events, the dictator of Burkholderia himself,

Selman Waksman, agrees to go to the UN headquarters to discuss a

potential arms reduction treaty

• After a mere 10 hours on American soil, Waksman is found dead in his

hotel room

• Enraged, the Burkholderian government threatens war against the

United States, which responds immediately by readying its military for

an engagement.

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The Suspects• In order to avert conflict, the NYPD Forensics

Unit promptly begins investigation of the crime scene

• Two blood samples are found on murder weapon

• Three suspects are placed under investigation: o Bethesda Waksman, the dictator’s wife

o Albert Lehninger, a CIA operative personally accused by the Burkholderian government

o Chuck Hegel, the US Secretary of Defense, the man who was last seen with Waksman by a third party

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The DNA Obtained• Blood samples from the three suspects:

o Victim’s Wife

o CIA Operative

o Secretary of Defense

• Two blood samples from the crime

scene

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What is DNA?• Genetic Material

o Responsible for diversity

o Each person (except identicaltwins) has a unique genome

• Double helix structure

• Unit is called a nucleotide

• 4 base pairso Adenine (A) : Thymine (T)

o Guanine (G) : Cytosine (C)

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Restriction Fragment Length

Polymorphisms (RFLPs)• DNA is variable, and RFLPs are one such variation.

• These variations are in the size of fragments that

exist between “restrictions sites”

• A restriction site is a short fragment of DNA that is

recognized and cut by a specific enzyme.

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…GAATTC……CTTAAG…

The Process1. DNA Extraction

2. Enzymatic Digestion

3. Gel Electrophoresis

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Step 1: DNA Extraction

• Cell Lysiso Detergent, salt, and water

• Solids Separation

• Precipitationo Ethanol

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Step 2: Enzymatic Digestion• Different restriction enzymes are added that

cut DNA at specific cut sites.

• Cut sites used for samples: EcoRI and PstI

• Added to solution:o Active enzymes

o Buffer solution

o DNA

o Water

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Step 3: Gel Electrophoresis• Combine digested DNA with dense dye

• Load samples into agarose gel wells

• Connect power supply and DNA will flow through

the gel from negative charge to positive chargeo DNA is separated by size

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Our DNA Samples

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MWLadder

Scene Blood

2

Scene Blood

1

Sec. of Defense

CIA Op.

WifeVictim

Simple Tandem Repeats(STRs)• Individuals have different number of repeated units

in certain areas of DNAo Individual 1: AATCAATCAATCAATCAATCAATCAATCAATCAATC

o Individual 2: AATCAATCAATC

• These areas of STR(loci) are targeted with sequence

specific primers and amplified using PCR.

TTAGTTAGTTAG

AATCAATCAATCAATCAATCAATCAATCAATCAATCAATCAATCAATCAATC

TTAGTTAG

This gives us fragments of different lengths for different individuals. These fragments are separated and detected using gel electrophoresis.

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How accurate?• Each STR allele will be shared around 5-20% of

individuals….

• Accuracy comes from looking at several STR loci

simulataneously

• 0.2*0.2*0.2*0.2*0.2*0.2 = .000064

-> 64/100000 people will be the same

The CODIS 13 core loci are used universally. Estimated

that probability of having the same 13 STRs is 1 in

10,000,000,000,000.

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Questions?

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Check us out at:

@CUGEM

http://igem.engineering.cornell.edu

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