Unit 2- DNA Analysis

Discovery of DNA structure

1950’s – Rosalind Franklin & Maurice Wilkins photograph DNA using x-ray diffraction

Discovery of DNA structure

1953 – James Watson & Francis Crickdevelop a model of a DNA molecule

DNA = Deoxyribonucleic Acid

DNA is located in the nucleus & in the mitochondria of cells

DNA is the code of genetic information that tells cells how and when to make proteins

Proteins make you unique = nose’s, toes, eyes, hair, your enzymes, etc.

Organization of DNA in cell Nucleus has 23

pairs of Chromosomes (46 total from parents)

Chromosomesare wound up strands of DNA

DNA sections that code for traits (aka proteins) are called genes

General Structure of DNA

Double helix — two coiled DNA strands

Composed of nucleotides pieces connected together

Four bases make the rungs: Adenine Cytosine Guanine Thymine

Bases always pair A to T and G to C (complementary base pairing)

In humans, the order of these bases is 99.9% the same.

Karyotype- picture of chromosomes

Human Genome Project (1990-2003)Goals: identify genes in

human DNA (20,000 +)

determine the sequences of ATCG’s that make up human DNA

store this information in databases

SPECIAL NOTE: DNA Analysis DOES NOT reveal personal information about a suspect… It cannot tell race, eye color, hair color, etc… not possible yet!

Bodily sources of DNAAll Cells that have a nucleus:

white blood cells (NOT RED BLOOD Cells- they don’t have a nucleus)

Semen saliva – contains buccal (cheek

cells) hair root teeth bone Any tissue

All cells contains thousands of mitochondriawhich contain maternal DNA

Uses of DNA Profiling identify potential

suspects

exonerate individuals

identify crime and casualty victims (9/11)

establish paternity

match organ donors

Milestones in DNA Analysis

Alec Jeffreys—1985 isolated DNA markers and called them DNA fingerprints

RFLP technologies used 1985- mid 1990’s

Kary Mullis—1985 developed PCR testing (perfected by the mid 1990’s)

1987- DNA evidence introduced for the first time in court

1988—FBI starts DNA casework

1991—first STR paper

1998—FBI launches CODIS database

Coding vs. Non- Coding sections of DNA

Coding DNA- 5 % of your DNA recipe that ―CODES‖ for proteins that make you unique

Junk DNA/ “Non Coding” DNA 95 % of DNA it doesn’t seem to code for anything (yet!). repeats the same base pair sequence over and over (aka Tandem Repeats)

Best for I.D’ ing people forensically… (b/c varies most in people)

Reading DNA fingerprints

•It’s Dino’s because it matches crime scene. •Bugsy has some DNA strands that aren’t in crime scene sample

Sometimes DNA is mixed at a crime scene

Miss Scarlet & Miss White each contributed

½ the DNA to the scene.

Paternity Case2paternity cases below (remember kids get ½ their DNA

from each parent):

Usually use terms: EXCLUDED or INCLUDED

DNA “Typing”

(Fingerprinting/ profiling/ analysis)

3 main technologies have been used:1) RFLP – Restriction Fragment Length Polymorphisms

- Developed in 1985 & used until mid 1990’s

2) PCR – Polymerase Chain Reaction- Developed in the 1980’s, but perfected in mid 1990’s- Still used today as a step in the STR process

3) STR- Short Tandem Repeats- Developed in the 1990’s - Used almost exclusively today

The RFLP Technique—Restriction Fragment Length Polymorphisms

Polymorphisms = Variations in DNA sequence between individuals

General Overview:

1) Isolate—separate DNA from the cell of desired individuals & evidence

2) Cut—using restriction enzymes to cut DNA into smaller fragments

3) Separate/ Sort— by size using electrophoresis which creates banding pattern.

4) Analyze—the specific allele patterns for identification

DNA Fingerprint

1. DNA is Isolated/ Extracted

This bead beater is used in the breaking apart or "lysing" of cells

3 Steps in a DNA extraction:a) Break open cells by grinding & remove

membrane lipids by adding a detergent.

b) Remove proteins bound to the DNA, by adding salt.

c) Precipitate DNA in cold alcohol (DNA is insoluble in alcohol and clings together)

DNA

2. Restriction enzymes cut DNA into fragments

Restriction Enzymes act as molecular scissors that cut DNA into pieces.

Come from bacterial cells where they were discovered

Each enzyme has a specific DNA sequence it will look for and cut.

Everyone has a lot of repeating DNA codes that are unique (tandem repeats)

Enzymes cut at these unique sections resulting in everyone having different fragments of DNA. (see image)

3. Gel electrophoresis- Separate fragments based on size

DNA is loaded into wells inside a gel An electrical current is moved through a gel (DNA is negatively

charged so it will move toward the positive side of the gel box)

molecules to sort by size. The smaller, lighter molecules will move the furthest on the gel, creating a banding pattern

Electrophoresis

1. Pipette & load

DNA into wells in

the gel.

2. Turn on electrical

supply to move DNA

3. See DNA “run” through gel

thanks to the electrical current

Small fragment=

fast

larger fragment=

slow

4. Radioactive markers are added to bond to the DNA to make it visible. Gels are “read”

Three Possible Outcomes

Match —The DNA profile appears the same. Lab will determine the frequency.

Excluded—The two samples originate from different sources.

Inconclusive—The data does not support a conclusion as to whether the profiles match.

Disadvantages of RFLP Fingerprinting

Requires lots of long pieces of DNA

Isssue:

longer pieces of DNA tend to degrade and break into smaller pieces, randomly.

Crime scene DNA is often in adverse conditions.

DNA Profiling Case-who’s the dad?

Surrogate mother

(her egg)

Surrogate’s husband

(is it his baby?)

Infertile couple she cannot conceive but he was a sperm donor

(is it his baby?)

DNA profiling Case- #2

“Joe Millionaire” &

potential father

Parents of father

Mother claiming infant son was fathered by

deceased “Joe Millionaire”

DNA Replication = copying 1 DNA molecule into 2 identical DNA molecules

Helicase = enzyme that unzips (breaks H Bonds) the strands of DNA.

DNA Polymerase = enzyme that attaches new complimentary bases on the unzipped strands

Semi-conservative Replication = new molecules are ½ new & ½ old strands

PCR— Polymerase Chain Reaction technique used for making

copies of a DNA molecule

valuable when the amount of evidence is minimal

Millions of copies of DNA can be made from a single speck of blood

Stems from knowledge of how DNA replicates itself

Thermocycler- machine used to do

PCR

PCR Procedure1) Heat DNA strands =strands

separate (unzip).

2) Cool the mixture and add a primer, a short sequence of base pairs, that will add to its complementary sequence on the DNA strand.

2) Add a DNA polymerase and a mixture of free nucleotides to the separated strands. DNA will re-build. Heat again to around 75 C for the completion.

Every cycle the DNA amount is doubled.

Advantages of PCR Typically 28- 32 PCR cycles are run resulting in

billions of copies of DNA (mathematically- 230)

Each cycle that doubles the DNA takes about 2 minutes.

Minute amounts of DNA may be used for amplification (less than 1 billionth of a gram).

Can get enough DNA from envelopes, stamps, soda cans, & cigarette butts to run PCR

STR- Short Tandem RepeatsThe basics

Current method of choice for DNA typing.

STR’s = short sequences of 2 to 5 bases that repeat themselves (100 -200 x’s)

RFLP’s (older technique) are 15- 40 base pairs long and can repeat up to 1000 times.

Example of STRs in a person

This is the TH01 loci for an individual

6 repeats from 1 parent

8 repeats from 1 parent

See “related internet links” on class website for STR demonstration.

By continuing the process with additional STRs from other genes, you can

narrow down the probability of DNA belonging to only one person. Here’s

all the ones that are used:

Short Tandem Repeats (STR) STR typing is visualized by peaks shown on a graph.

Each represents the size of the DNA fragment.

The possible alleles are numbered for each loci.

Norma's genotype is 15 repeats, 15 repeats at the locus D3S1358,

14 repeats, 16 repeats at vWA,

24 repeats, 25 repeats at FGA

(gets on from each parent)

Great website: http://www.genetica.com/GeneticaWebV2.nsf/XReadingtheResultsTechnicalInformation.xsp

EX: STRS Paternity Profile

Using STR DNA Profiling ResultsDavid & Karen are parents of a missing child:

DNA Profile from remains found in a shallow grave:

Could this be their child?

No, the DNA for vWA & FGA doesn’t show ½ from each parent

Short Tandem Repeats (STR) Procedure

Extract the gene TH01 from the sample. (TH01 has seven human variants with a repeating sequence of A-A-T-G)

Amplify the sample by means of PCR

Separate by electrophoresis

Examine the distance the STR migrates to determine the number of times TH01 repeats

Each person has two STR types for TH01—one inherited from each parent

This is the TH01 loci for an

individual:

Received 6 repeats from one parent

and 8 repeats from the other.

Combines the strengths of PCR and RFLP while minimizing the weaknesses:

requires small amount of DNA- need only 18 DNA bearing cells. 1 billionth of a gram (1/100th needed for RFLP).

STR’s found in great abundance in human genome so allow for greater discrimination in results (1 in 1 trillion people for some odds)

DNA is less susceptible to degradation because of the small amount needed.

Requires less time to run (12 hours in Saddam case)

Advantages of STR

DNA Identification- Product Rule for Probability

probability of a person matching a random DNA sample at any 1 STRS site is roughly 1/10

3 STRS sites? – 1/10 x 1/10 x 1/10 = 1/1000

all 13 STRS sites would mean that the chances of matching a random DNA sample are about 1 in 10 trillion:– 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10

x 1/10 x 1/10 x 1/10 x 1/10 x = 1/10,000,000,000,000

probability of two different people matching at all 13 STRS sites is virtually zero.

Types of DNANuclear

found in the nucleus inherited from both

parents each cell contains only

1 nuclei

Mitochondrial found in the cytoplasm inherited only from

mother each cell contains 100’s -

1000’s of mitochondria used when nuclear DNA

typing is not possible (old, degraded samples)

More costly, time consuming to test

Not very discriminating

FBI’s CODIS DNA Database

Computer system has:

Convicted Offenders DNA

Crime Scene DNA

Arrestee DNA

Unidentified/ Missing person’s DNA

Combined DNA Index System (1998)

System can cross reference info. from all 50 states• Prop 69 in CA (all felons or people who attempted to commit must have DNA taken)

Collection and Preservation of Biological Samples

Photograph objects containing samples

Limit personal contact & change gloves often

Disposable tweezers thrown out after each item

All clothing should go to lab, even if blood is not seen

Package items separately in breathable container (not plastic!)

blood is allowed to dry firstRefrigerate evidence until delivered to a lab

Evidence Source of DNA

•baseball bat or similar weapon sweat, skin, blood, tissue

•hat, bandanna, or mask sweat, hair, dandruff

•eyeglasses sweat, skin

•facial tissue, cotton swab mucus, blood, sweat, semen, ear wax

•dirty laundry blood, sweat, semen

•Toothpick, cigarette, stamp or envelope, bite mark

saliva

•tape or ligature skin, sweat

•bottle, can, or glass saliva, sweat

•used condom semen, vaginal/ rectal cells

•blanket, pillow, sheet sweat, hair, semen, urine, saliva

•―through and through‖ bullet blood, tissue

•Fingernail (or partial) blood, sweat, tissue

Cover Photo Controversy

Time was forced to recall the original cover story (left) and replace it with the one on the right.