Download Tuesday 4/8/14

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 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”
5’-GGATCC-3’
Bam H1 site:
3’-CCTAGG-5’
Restriction Enzymes Pt 1 YouTube
Enzyme Site
Recognition
Restriction site
Palindrome
• Each enzyme digests
(cuts) DNA at a
specific sequence =
restriction site
• Enzymes recognize
4- or 6- base pair,
palindromic
sequences
(eg GAATTC)
Fragment 1
Fragment 2
Enzyme cuts
5 vs 3 Prime
Overhang
• Generates 5 prime
overhang
Common Restriction
Enzymes
EcoRI
– Eschericha coli
– 5 prime overhang
Pstl
– Providencia stuartii
– 3 prime overhang
Restriction enzymes
Enzyme
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 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.
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 Blot
January 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
January 28, 2003
Southern blot
General Scheme for Southern Blot
Restriction Digest
Gel Electrophoresis
DNA Preparation:
Denaturation/Depurination
Transfer to filter: Blotting
Detecting DNA: Probing
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 rapemurder 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