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DNA

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.

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DNA

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  1. 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

  2. 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

  3. 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

  4. 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

  5. DNA RFLP typing process

  6. 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

  7.  Variants of STR TH01 Top DNA strand-six repeats of sequence A–A–T–G Bottom DNA strand-eight repeats of sequence A–A–T–G

  8. http://wps.prenhall.com/wps/media/objects/3801/3892550/DNACD_mod07-2-08.swfhttp://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

  9. Process of DNA Typing • DNA Extraction • DNA Purification • DNA Quantitation • DNA Amplification-PCR • Fragment Analysis-Capillary Electrophoresis • Evaluation-Electropherogram

  10. DNA Extraction and Purification • Extraction of DNA Sample-remove DNA from cells-DNA Extraction Machine • Purification-Separate DNA from contaminants

  11. Portable Genetic Analyzer

  12. 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.

  13. 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

  14. 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

  15. PCR

  16. Thermalcycler-A PCR MachineThe PCR Songhttp://bio-rad.cnpg.com/lsca/videos/ScientistsForBetterPCR/

  17. 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,2-8 http://www.sumanasinc.com/webcontent/animations/content/pcr.html

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

  19. PCR Animations http://www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html http://www.dnalc.org/ddnalc/resources/animations.html

  20. 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

  21. Capillary 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 • 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

  22. Capillary Electrophoresis

  23. 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

  24. 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

  25. DNA 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

  26. Electropherogram

  27. 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

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

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

  30. Who’s your Daddy?Paternity testing • ABO blood typing-exclude paternity, but cannot confirm paternity • DNA testing can confirm paternity

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

  32. The Y Chromosome

  33. 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

  34. Every cell -hundreds of mitochondria Each mitochondrion-many copies of DNA Differences between individuals in mtDNA-two specific segments HV1 and HV2

  35. 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/

  36. 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

  37. 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

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