DNA Evidence
Scientific Breakthrough The discovery of the structure of DNA, coupled with a new understanding of how it is replicated and transcribed from gene to protein may be the single most amazing discovery of the 20th century! Applying this new knowledge to forensics wasn’t apparent right away…
History 1985 – Alec Jeffreys et.al. @ Leicester University While investigating a human gene, they discovered that portions of the DNA structure of certain genes are unique to each individual. DNA Fingerprinting/Typing was developed
Forensic Scientists jumped at the opportunity to turn this biological evidence from class to Individual Courts now accept DNA evidence as reliable
DNA Refresher There are 60 trillion cells in the human body Chromosomes – rod-like structures made up of DNA that is coiled around proteins Human DNA contains 3 billion base pairs approximately 25,000 genes Genes instruct the body cells to make proteins that do anything including structural proteins to make new cells, enzymes, determine hair and eye color, etc.
Complementary Base-pairing DNA Refresher DNA is a polymer (a string of many molecules put together) The pieces of the string are nucleotides Structure: Double helix Sugar phosphate backbone Joined together by A T C G Complementary Base-pairing A-T G-C
DNA Refresher - Replication DNA Helicase binds to the DNA strand and causes the helix to unwind and the 2 strands break apart With the 2 strands separated there is now a template strand for free nucleotides in the nucleus to attach DNA Polymerase is an enzyme that matches up the free nucleotides with their complementary base on the template strand of DNA DNA Polymerase is also the proofreader – checking for errors in base pairing and repairing as needed
DNA Fingerprint Bozeman Science DNA Fingerprinting Video: https://www.youtube.com/watch?v=DbR9xMXuK7c
Human DNA is approximately 3 billion base pairs long, and we have about 30,000 different genes. In order to study a specific gene we need to cut down the long strand of DNA and select only the specific gene or region that we are working on Restriction Enzymes = “molecular scissors” Image source: Microsoft Clipart Gallery
Cuts can be ‘blunt’ or ‘sticky’ Restriction Enzyme – Eco R1 Recognition Site ACTGGTACGAATTCCGTA TGACCATCCTTAAGGCAT AATTCCGTA GGCAT ACTGGTACG TGACCATCCTTAA Sticky Ends! Sticky ends will bind to complementary bases that come along, making them useful
Once DNA has been cut into fragments by restriction enzymes, the fragments can be sorted or separated by size How?
Gel Electrophoresis An electrical current is used to separate a mixture of different fragments of DNA DNA Sample Negative electrode Positive electrode
Gel Electrophoresis Because DNA has a negative charge, the fragments move toward the positive electrode Pores in the gel allow small molecules to move through the gel quickly, while large molecules get “stuck” and move slowly
Gel Electrophoresis So, the length of a DNA fragment can be estimated compared to other fragments by measuring how far each fragment migrated through the gel DNA fragments appear as different bands or lines on the gel (after staining)
Restriction Maps The band pattern can be thought of as a map of the original strand of DNA The map shows the lengths of DNA fragments between restriction sites on a DNA strand
How can we get a large enough sample of DNA to work with? Polymerase Chain Reaction (PCR) 1 Cycle 2 Cycles 3 Cycles
Polymerase Chain Reaction (PCR) A technique that produces millions (or billions!) of copies of a specific DNA sequence in hours Invented by Kary Mullis in 1983 He patented the process and sold the rights for $300 million Won the Nobel Prize in Chemistry in 1993 Under the right set of conditions, DNA polymerase enzymes will make new copies of DNA in a test tube, just like they do in your cells! Sample size is no longer an issue in investigations!
Polymerase Chain Reaction (PCR) 4 Starting Materials: DNA strand DNA polymerase Nucleotides Primers Separating Tube with all reactants is heated to over 90C to separate the strands of DNA Binding Tube is cooled to 55C, primers bind to DNA Copying Tube heated to 72C, polymerase binds new nucleotides until segment is copied
Polymerase Chain Reaction (PCR) Each PCR cycle doubles the number of DNA copies 1 > 2 > 4 > 8 > 16 > 32 > 64 > 128 > 256 > 512… After 30 cycles, more than 1 billion copies! What is a primer? A short segment of DNA that acts as a starting point for the new strand
DNA Fingerprinting – a restriction map! Our DNA is 99.9% identical, how do we find the differences? The greatest differences in DNA sequence between people are in the non-coding sections of our DNA (introns) So….DNA fingerprinting focused on the introns, which often include stretches of nucleotide sequences that repeat several times Each person’s DNA differs in how many repeats they have
DNA Fingerprinting – a restriction map! DNA fingerprinting has become reliable and widely used in US courts since the 1990s In most forensic cases, at least 5 regions of DNA are analyzed This technique can be used to: identify perpetrators of a crime identify fathers in paternity cases identify unknown remains clear persons wrongly accused identify crime and catastrophe victims match organ donor with recipients in transplant programs