Wednesday 4/9/14

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 343-348.

questions 9-11 pages364-365

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 WorldApplications

• 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

ProceduresDay One

DNA DigestionTemperature 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

ProceduresDay Two

DNA Fingerprinting

ProceduresDay Three

Electrophoresis Analysis of Stained Gel

Determinerestriction fragmentsizes

• DNA marker

• Measure distance traveled by restriction fragments

• Determine size of DNA fragments

Identify the relatedsamples

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 enzymesCleave or cut DNA

1962: “molecular scissors” discovered in in bacteriaE. coli bacteria have an enzymatic immune system that recognizes and destroys foreign DNA3,000 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 enzymesEnzyme Sequence cut

BAM HI GGATCC Between G and G

Hae III GGCC Between G and C

Pst I CTGCAG Between A and G

Bgl II AGATCT Between C and T

Restriction Endonucleases

Restriction enzyme animation

http://www.dnai.org/b/index.html

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 PO4)

• 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 353-355. 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 357-360. answer questions 16-18 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 filedby 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.

January 28, 2003Southern Blot

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

January 28, 2003Southern Blot

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