Title: DNA DeoxyRibonucleic acid
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2DNA
- 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-doesnt directly code for
protein-Junk DNA
3Polymorphisms
- 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
4Techniques 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
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6DNA 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
7DNA RFLP typing process
8Intertwined strands of DNA representing segments
of two chromosomes Chromosome segment on the
left contains three repeating sequences of
TAG Chromosome segment on right has two
repeating sequences of TAG
9 Variants of STR TH01 Top DNA strand-six repeats
of sequence AATG Bottom DNA strand-eight
repeats of sequence AATG
10- http//wps.prenhall.com/wps/media/objects/3801/389
2550/DNACD_mod07-2-08.swf - http//wps.prenhall.com/wps/media/objects/3801/389
2550/DNACD_mod02-1-12.swf - http//wps.prenhall.com/wps/media/objects/3801/389
2550/DNACD_mod02-1-13.swf
11Process of DNA Typing
- DNA Extraction
- DNA Purification
- DNA Quantitation
- DNA Amplification-PCR
- Fragment Analysis-Capillary Electrophoresis
- Evaluation-Electropherogram
12DNA Extraction and Purification
- Extraction of DNA Sample-remove DNA from
cells-DNA Extraction Machine - Purification-Separate DNA from contaminants
13Portable Genetic Analyzer
14Quantitation
- 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.
15RT-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
16DNA 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
17PCR
18Thermalcycler-A PCR Machine The PCR Song
http//bio-rad.cnpg.com/lsca/videos/ScientistsForB
etterPCR/
19Automated 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,2-8
- http//www.sumanasinc.com/webcontent/animations/co
ntent/pcr.html
20Appropriate primers flanking the repeat units of
a DNA segment must be selected and put in place
in order to initiate the PCR process.
21PCR Animations
- http//www.dnalc.org/ddnalc/resources/shockwave/pc
ranwhole.html - http//www.dnalc.org/ddnalc/resources/animations.h
tml
22Genetic 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
23Capillary Electrophoresis
- Fragment 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 - STRs 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/3801/389
2550/DNACD_mod04-2-08.swf
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25Capillary Electrophoresis
26Genetic 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
27Data 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
28DNA Analysis
- Data 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
29Electropherogram
30Process 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/3801/389
2550/DNACD_mod04-2-11.2.swf
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33http//wps.prenhall.com/wps/media/objects/3801/389
2550/DNACD_mod04-2-11.2.swf
34CODIS
- Combined DNA Index System-database of DNA from
felons and crime scenes
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36Whos your Daddy?Paternity testing
- ABO blood typing-exclude paternity, but cannot
confirm paternity - DNA testing can confirm paternity
37Y-STRs
- http//wps.prenhall.com/wps/media/objects/3801/389
2550/DNACD_mod08-4-01.swf
38The Y Chromosome
39Whos 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
40Every cell -hundreds of mitochondria Each
mitochondrion-many copies of DNA Differences
between individuals in mtDNA-two specific
segments HV1 and HV2
41- DNA Evidence Collection at Crime Scene
Interactive - http//media.pearsoncmg.com/ph/chet/chet_saferstei
n_hsforensics_1/cw_le_dna/beg/burglary/burglary_01
.htm - http//media.pearsoncmg.com/ph/chet/chet_saferstei
n_hsforensics_1/cw_le_dna/adv/burglary/burglary_01
.htm - Gel Electrophoresis interactive simulation
- http//learn.genetics.utah.edu/units/biotech/gel/
42STR Profiling Sensitivity
- STR Profiling-can detect 125 picograms
- 1 picogramone-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
43Amounts of DNA
- RFLP/VNTRs- 50 ng-1000 ng 1985-1995
- PCR/STRs- 0.5-2 ng 1991 - present
- LCN/STRs lt0.1 ng 1999-present