DNA

• Three billion base pairs in DNA• Human cells-23 pairs of chromosomes

except gametes and rbcs• 99.5% similarity among individuals• Genome-total DNA in the cell• 5% of genome is encoded-directs

synthesis of proteins• 95% is non-encoded-doesn’t directly

code for protein-Junk DNA

Polymorphisms

• Vary in length (length polymorphism) and base sequence

• Two sequences:1. Variable Number Tandem Repeats

(VNTRs)-same base sequence repeats throughout a specific locus-can be hundreds of base pairs

2. Short Tandem Repeats (STRs)-3-7 bp long-repeat throughout loci-ex.

AGTT AGTT AGTT AGTT AGTT AGTT

Techniques of DNATyping

1. Restriction Fragment Length Polymorphism (RFLP)-used restriction enzymes to cut DNA into fragments-individuals differ in where these enzymes cut-different fragment sizes based on VNTRs

2. Polymerase Chain Reaction (PCR)-STR containing regions amplified

DNA Fingerprinting

• Alec Jeffreys-1985-areas of DNA exhibit polymorphism using RFLPs

• These polymorphic areas allow scientists to identify individuals

• DNA Fingerprinting-process of isolating and analyzing the DNA

DNA RFLP typing process

Intertwined strands of DNA representing segments of two chromosomes Chromosome segment on the left contains three repeating sequences of T–A–G

Chromosome segment on right has two repeating sequences of T–A–G

 Variants of STR TH01Top DNA strand-six repeats of sequence A–A–T–GBottom DNA strand-eight repeats of sequence A–A–T–G

• http://wps.prenhall.com/wps/media/objects/3801/3892550/DNACD_mod07-2-08.swf

• http://wps.prenhall.com/wps/media/objects/3801/3892550/DNACD_mod02-1-12.swf

• http://wps.prenhall.com/wps/media/objects/3801/3892550/DNACD_mod02-1-13.swf

Process of DNA Typing

1. DNA Extraction2. DNA Purification3. DNA Quantitation4. DNA Amplification-PCR5. Fragment Analysis-Capillary

Electrophoresis6. Evaluation-Electropherogram

DNA Extraction and Purification

• Extraction of DNA Sample-remove DNA from cells-DNA Extraction Machine

• Purification-Separate DNA from contaminants

Portable Genetic Analyzer

Quantitation

• Quantitation-Determine amount of DNA in the sample

• QuantiBlot quantitation-determines amount of DNA extracted from original sample-hybridization probes and colorimetric or chemiluminescent detection.

RT-PCR• RT PCR amplifies DNA sample and studied during real time, or

while it happens. • Data is collected during the process as it analyzes changes of

amount of fluorescence from DNA amplification• TaqMan Probes-labeled with two fluorescent dyes sensitive to

different wavelengths-hybridize to DNA that is amplified.-probe checks for changes in fluorescence caused by displacement of double-dyed probe from a specific area in amplified DNA.

• Quantitation of DNA and determines condition of DNA present• Amplifies that DNA.• Real Time PCR is more sensitive than Quantiblot-risks of

contamination are higher• The Quantiblot system-10 to 0.15 ng of DNA• RT PCR -from 0.003 to more than 50 ng/microliter of DNA • 1 nanogram= one billionth of a gram

DNA Amplification-PCR• Polymerase chain reaction (PCR)-make copies of

loci with STRs-makes enough DNA to be detected • The DNA sample is denatured, cooled, primers

annealed, free nucleotides synthesize complementary strands of DNA

• This process is repeated for about 28 cycles • Taq DNA polymerase-attaches nucleotides to DNA • DNA primers initiate replication• PCR rxns include template DNA which will be

amplified and deoxynucleotide triphosphates that will form the new DNA strands

PCR

Thermalcycler-A PCR Machine The PCR Song

http://bio-rad.cnpg.com/lsca/videos/ScientistsForBetterPCR/

Automated Process of PCR

a. Heated to 95o C- Denature (separate) DNA into two strands

b. DNA primers hybridize to complementary bases

c. DNA Polymerase binds to primer and adds bases of DNA

Process repeats-one cycle-4 strands,2-8http://www.sumanasinc.com/webcontent/animations/content/pcr.html

Appropriate primers flanking the repeat units of a DNA segment must be selected and put in place in order to initiate the PCR process.

PCR Animations

http://www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html

http://www.dnalc.org/ddnalc/resources/animations.html

Genetic Analyzer

• Fluorescent Detection• Charge-coupled device-CCD camera

detector monitors 525-625 nm wavelengths

• Photons fall on pixels-silicon of CCD absorbs energy and converts light into electronic charge

• Number of electrons collected-depends on light level, exposure, and wavelength

• Separates STR regions by size-laser measures fluorescent emissions

Capillary ElectrophoresisFragment Analysis- Electrophoresis- electrical current is passed

through polymer -DNA moves toward positive end and separates into fragments by size - Acrylamide polymer (capillary) electrophoresis

• Sixteen capillaries used simultaneously- run 16 different samples in 45 minutes

• STR’s lengths differ among individuals• Smaller STRs move through capillaries faster• Different loci-different colored fluorescence-detected by laser • Profiles show # of repeats at a locus by comparison of where

fluorescence occurred in sample to a standard ladder ABI Prism 3130 Genetic Analyzer-uses capillary electrophoresis-

separates STR segments by size and measures them with laser by recording light fluorescence

http://wps.prenhall.com/wps/media/objects/3801/3892550/DNACD_mod04-2-08.swf

Capillary Electrophoresis

Genetic Analyzer• Alleles represents as peaks• Software-Applied Biosystems(ABI) GeneScan

and GenoTyper • GeneScan identifies signal corresponding to

dyes in sample • Uses GeneScan analysis-identify & label peaks• Electropherogram-Graph representing STRs

Data Analysis

• Isolate locus of DNA and determine number of STRs of given sequence

• Individuals receive different numbers of repeats from each parent

• Thirteen loci-probability of two individuals with same STRs-one in several hundred trillion

DNA AnalysisData Analysis• GeneMapperIDTM v3.2 software-Applied

Biosystems-computer program that automatically defines and assigns loci according to a set a standard rules

• DNA analyst manually assesses them• The success of DNA profiling is determined

by total peak heights, total peak area, and overall quality of DNA profiles

Electropherogram

Process of DNA Typing

Evaluation-Computer analyzes data-Electropherogram-stored on CD as evidence-compared with suspect DNA profile or entered into CODIS

 http://wps.prenhall.com/wps/media/objects/3801/3892550/DNACD_mod04-2-11.2.swf

http://wps.prenhall.com/wps/media/objects/3801/3892550/DNACD_mod04-2-11.2.swf

CODIS

• Combined DNA Index System-database of DNA from felons and crime scenes

Who’s your Daddy?Paternity testing

• ABO blood typing-exclude paternity, but cannot confirm paternity

• DNA testing can confirm paternity

Y-STRs

• http://wps.prenhall.com/wps/media/objects/3801/3892550/DNACD_mod08-4-01.swf

The Y Chromosom

e

Who’s your momma?Mitochondrial DNA

• Passes through maternal lineage• Hair shaft, teeth, old bones• Is very stable• Inherited unchanged from mother• All cell components of developing zygote

come from mother• All cells in body contain identical DNA• mtDNA mutation-once every 6,500 years• Used in hair analysis-only living part is

follicle-can get nuclear DNA there-but if hair is cut-can get mtDNA from dead cells

Every cell -hundreds of mitochondriaEach mitochondrion-many copies of DNA Differences between individuals in mtDNA-two specific segmentsHV1 and HV2

• DNA Evidence Collection at Crime Scene Interactive

• http://media.pearsoncmg.com/ph/chet/chet_saferstein_hsforensics_1/cw_le_dna/beg/burglary/burglary_01.htm

• http://media.pearsoncmg.com/ph/chet/chet_saferstein_hsforensics_1/cw_le_dna/adv/burglary/burglary_01.htm

• Gel Electrophoresis interactive simulation• http://learn.genetics.utah.edu/units/biotech/gel/

STR Profiling Sensitivity

• STR Profiling-can detect 125 picograms

• 1 picogram=one-trillionth of a gram• One cell has 7 picograms of DNA• 18 DNA bearing cells are needed to

get a STR profile• Low Copy Number (LCN) DNA-less

than 18 DNA bearing cells

Amounts of DNA

• RFLP/VNTRs- 50 ng-1000 ng 1985-1995

• PCR/STRs- 0.5-2 ng 1991 - present

• LCN/STRs <0.1 ng 1999-present