Wednesday 4/9/14 AIM: How is DNA useful to forensic science? DO NOW: List at least 4 sources of DNA that could be extracted from a crime scene HOMEWORK: Text read pages questions 9-11 pages

DNA: deoxyribonucleic acid It is a polymer Big molecule made of repeating subunits DNA is a chain of Nucleotides a nucleotides is made of three parts: a phosphate, a nitrogenous base, and a 5 carbon sugar.

Forensic source of DNA Blood and bodily fluids are the most common sources Which part of blood gives us DNA? Leokocytes: White blood cells A single drop of blood may contain 7,000 to 25,000 white blood cells DNA fingerprint or profile

DNA fingerprint Identify the potential suspect Clear wrongfully suspected person Identify crime and victims Establish paternity and family relationships Match organ donors

Wednesday 4/9/14 AIM: How is DNA manipulated in the Forensics lab? DONOW: Explain how you would separate a blood sample to extract DNA HW: Page 364 q 1-3 Last night was 9-11

Preparing DNA for analysis Isolate or remove DNA from sample ex: skin, clothing, weapon Extract DNA from the cells (centrifuge) Enzymes are then used to isolate DNA from chromosome Enzyme: protein catalyst: causes a chemical reaction that may not ordinarily take place

DNA profiling, testing, typing or genetic fingerprint Process that identifies individuals based on their individual DNA

DNA Fingerprinting Real World Applications Crime scene Human relatedness Paternity Animal relatedness Anthropology studies Disease-causing organisms Food identification Human remains Monitoring transplants

Collect Buccal Cells

Steps to making a DNA fingerprint 1- Extract DNA from cell nucleus 2- Add restriction enzymes to cut DNA into pieces 3- Separate fragments with gel electrophoresis 4- Make a copy of results using southern blot 5- add radioactive DNA probe to visualize 6- visualize fragments and analyze results

Forensic DNA Fingerprinting: Using Restriction Enzymes

DNA Fingerprinting Procedures Day One

DNA Digestion Temperature Why incubate at 37°C? Body temperature is optimal for these and most other enzymes What happens if the temperature is too hot or cool? Too hot = enzyme may be denatured (killed) Too cool = enzyme activity lowered, requiring longer digestion time

DNA Fingerprinting Procedures Day Two

DNA Fingerprinting Procedures Day Three

Electrophoresis Analysis of Stained Gel Determine restriction fragment sizes DNA marker Measure distance traveled by restriction fragments Determine size of DNA fragments Identify the related samples

Thursday 4/9/14 AIM: how are DNA fragments separated? DO NOW: What is the function of a restriction enzyme? 2 minute mystery Big dipper Homework: Textbook page 365 q 17

Why would you use restriction enzymes in a forensics lab? To cut up DNA samples and create a DNA fingerprint to identify a piece of evidence

Would you make an arrest based on the evidence below?

Restriction Fragment Length Polymorphism R:Restriction: enzymes are used to cut the DNA F-fragments: creates many pieces of DNA L-length of each fragment varies among individuals P-polymorphisms: greek term meaning many shapes

Restriction Endonucleases Also called restriction enzymes Cleave or cut DNA 1962: “molecular scissors” discovered in in bacteria E. coli bacteria have an enzymatic immune system that recognizes and destroys foreign DNA 3,000 enzymes have been identified, around 200 have unique properties, many are purified and available commercially

Pd 3 Friday 4/11/14 AIM: how can we separate DNA fragments? DO NOW: how were restriction enzymes first found?

Restriction Endonucleases Recognition sites have symmetry (palindromic) “Able was I, ere, I saw Elba” Bam H1 site: 5’-GGATCC-3’ 3’-CCTAGG-5’

Restriction Enzymes Pt 1 - YouTube

Enzyme Site Recognition Each enzyme digests (cuts) DNA at a specific sequence = restriction site Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC) Palindrome Restriction site Fragment 1 Fragment 2

5 vs 3 Prime Overhang Generates 5 prime overhang Enzyme cuts

Common Restriction Enzymes EcoRI – Eschericha coli – 5 prime overhang Pstl – Providencia stuartii – 3 prime overhang

Restriction enzymes EnzymeSequencecut BAM HIGGATCCBetween G and G Hae IIIGGCCBetween G and C Pst ICTGCAGBetween A and G Bgl IIAGATCTBetween C and T

Restriction Endonucleases Restriction enzyme animation

NOW WHAT … Separate the fragments

Gel electrophoresis The standard method for separating DNA fragments is electrophoresis through agarose gels.

Terms Agarose is a polysaccharide (carbohydrate) polymer material, generally extracted from seaweed. Gel electrophoresis is a method that uses an electrical current and a gel matrix to separate molecules like DNA and proteins. Buffer a solution containing either a weak acid and its salt or a weak base and its salt, which is resistant to changes in pH.

Agarose Gel Electrophoresis Agarose gels must be prepared and run in a buffer containing ions. Ions are charged particles (like those found in salt) and are necessary to carry a charge A buffer is a substance that resists changes in pH. – It will neutralize a base---make it into water – It will neutalize an acid---make it into water

Agarose Gel Electrophoresis Buffers prevent the pH from changing by reacting with the H+ or OH- products Most common buffer used is called TRIS – [tris(hydroxymethyl)aminomethane]

Agarose Gel Electrophoresis After RFLP, DNA is applied to a slab of gelled agarose The sample is loaded with a loading buffer—containing dyes and glycerol or sugar Electric current is applied across the gel DNA is negatively charged (due to PO 4 ) Migrates from the negative (black) electrode to the positive (red) electrode

Like charges repel Rate of migration of DNA through agarose depends on the size of DNA Smaller DNA fragments move more quickly

Friday 4/11/14 AIM: how can we visualize a DNA fingerprint? DO NOW: explain how DNA fragments are separated in a gel electrophoresis

DO NOW Answer Add electric current to the fragmented DNA The negative current repels the negative DNA Smaller fragments move faster through the gel Larger fragments remain close to the top

Remember Restriction enzymes cut and leave a single strand of DNA open for complementary bonding

Wednesday 4/23/14 AIM: How can we replicate DNA for further analysis? DO NOW: If everyone has the sameA,T,C and G nucleotides, then how are the billions of people different? HW: Text read pages answer questions 12 and 13 on page 365

PCR: Polymerase chain reaction Copies DNA used to make many copies of a gene after isolation Allows the analysis of short pieces of DNA or RNA without having to clone it

Polymerase chain reaction Uses a change in temperature Ingredients – Uses heat resistant bacteria such as archaebacteria – Sample of DNA with desired gene – 4 different types of individual nucleotides – 2 short sequences of complementary primers

Steps in PCR Step 1: heat DNA at 94 degrees to denature the double helix (break the H bonds between complementary base pairs) Step 2: cool the mixture to about 64 degrees and add a DNA primer DNA primer: short sequence of nucleotides that is complementary to the gene sequence you want to copy

Step 3; heat to 74 degrees add DNA polymerase and A,T,C,G DNA polymerase causes complementary base pairing This is the step that actually is MAKING or REPLICATING the DNA

PCR facts There are 3 billion letters in the DNA code of each cell PCR makes copies of pieces of that code NOT THE WHOLE THING It takes 2 minutes for the process to be complete It takes 3 hours to make a million copies PCR requires 50% less DNA than RFLP

Polymerase Chain Reaction (PCR) - YouTube

Thursday 4/24/14 AIM: how does DNA connect a suspect to a crime? DO NOW: Why do forensic scientists analyze DNA? HOMEOWRK: Text read pages answer questions pg 365

DNA Evidence DNA evidence-has many uses within the legal system and criminal cases. – Proving someone guilty or innocent for a crime they have or have not committed. – Identification – Paternity Testing First criminal identification card filed by the NY State Bertillon Bureau

Criminal Cases DNA evidence has exonerated people accused of committing crimes. Only about 30% of all DNA tests run by the FBI have exonerated an accused person DNA evidence is still not as useful as fingerprinting.

Southern BlotJanuary 28, 2003 Identification Used to determine the sex, race, or even name of unnamed victims of crimes. Used in military to identify those who have died in battle, similar to the purpose of dog tags. Typical dog tags

Southern BlotJanuary 28, 2003 Paternity Testing Evidence can be used to compare the DNA of the suspected parent(s) and that of the child and determine the real parent.

Southern blot

General Scheme for Southern Blot Gel Electrophoresis DNA Preparation: Denaturation/Depurination Transfer to filter: Blotting Detecting DNA: Probing Restriction Digest

Southern Blot

DNA probe: identify specific genes Short single strand sequence of DNA labeled with a radioactive isotope, dye, or enzyme used to locate a particular nucleotide sequence or gene on a DNA molecule Once located,the gene can then be isolated Radioactivity of probe produces dark spots on top of genes of interest

Forensic history of DNA analysis 1986: suspect was exonerated for double rape- murder in england 1987: first time DNA ID is used to establish familial relationship between Ghanaise boy and his mother in United Kingdom 1994: husband convicted of ex wife murder on Prince Edward Island Canada due to cat hair DNA analysis NFL placed synthetic DNA on footballs used in super bowl XXXIV to prevent memorabilia fraud

The Innocence Project 1992 Cardozo School of Law 314 people exonerated 172 of those cases were assisted by Innocence Project After more than 17 years in prison Eddie Joe Lloyd who was convicted of the rape and murder of a 16 yo girl in Michigan was pardoned and released In August 2002

CODIS Combined DNA Index System Analyses Short Tandem Repeat (STR)sequences of DNA – Locations on chromosomes that repeat specific sequence of 2-10 base pairs Analyzes Variable number of Tandem repeats (VNTR) – Identifies repeats of 9-80 base pairs Forensic scientists scan 13 DNA regions from person to person then create a DNA profile CODIS looks at 13 specific STRs

Assessment In your own words explain how DNA analysis is used in our world

Tuesday 4/22/14 AIM: how can we connect a suspect to a crime using RFLP and gel electrophoresis? DO NOW: How do restriction enzymes work? What are DNA palindromes? HOMEWORK:

Restriction Fragment Length Polymorphisms

Restriction enzymes Recognize specific sequences of DNA and cut them into pieces Palindrome: has the same DNA sequence front and back EX: GGTACC CCATGG

RFLP Restriction Fragment Length Poymorphism It is a process used in the beginning of a DNA fingerprint Specifically it cuts DNA into pieces Each fragment will be separated based on its sized by the process of gel electrophoresis Restriction enzymes: recognize specific sequences called palindromes Palindrome: same nucleotide sequence front and back

Agarose Gel Electrophoresis