Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Forensic dna typing by John M Butler


Published on

First 4 chapter slides. Notes on First 4 chapters

Published in: Education, Technology
  • Be the first to comment

Forensic dna typing by John M Butler

  1. 1. Forensic DNA Typing By JOHN M.BUTLER
  2. 2. Muhammad Ahmad Bachelor of Science in Bioinformatics OMSATS Institute of Information Technology COMSATS Road, off G. T Road, Sahiwal.
  3. 3. Chapter 1 Overview and History of DNA Typing ©2002 Academic Press
  4. 4. History of forensic DNA analysis  Described by Dr.Alec Jeffrey  There are repeated regions over and over again next to each other.  The number of repeated regions differ from individual to individual.  These regions are known as VNTRs.  RFLP is used to examine VNTRs.
  5. 5. Comparison of DNA Typing Methods  The efficiency of methods depends on 1. Ability to differentiate two individuals 2. Speed with which results are Obtained
  6. 6. Comparison of DNA Typing Techniques Speed of Analysis (Technology) Power of Discrimination (Genetics) Low High Slow Fast Markers Used (Biology) RFLP Single Locus Probes RFLP Multi-Locus Probes ABO blood groups Multiplex STRs DQ single STR D1S80 mtDNA PolyMarker ©2002 Academic Press
  7. 7. mtDNA Importance  As in previous shown quadrant mtDNA has lowest efficiency  But it can be very helpful in cases where severely DNA samples are involved or in maternally related cases.
  8. 8. Sample Obtained from Crime Scene or Paternity Investigation DNA Extraction DNA Quantitation PCR Amplification of Multiple STR markers Biology Separation and Detection of PCR Products (STR Alleles) Technology Sample Genotype Determination Genetics Comparison of Sample Genotype to Other Sample Results If match occurs, comparison of DNA profile to population databases Generation of Case Report with Probability of Random Match Steps in Sample Processing
  9. 9. Separation and Detection Methods 1. Separation Methods I. Slab gel II. Capillary Electrophoresis: used to separate ionic species by their charge and frictional forces and hydrodynamic radius 2. Detection methods Fluorescence detection methods are used ABI Prism 310 Genetic Analyzer
  10. 10. Chapter 2 DNA BIOLOGY REVIEW
  11. 11. Basic Concept  DNA is our genetic blue- print .  DNA provides a computer program that determines our physical features and many other attributes.  The entire DNA in a cell, is referred to collectively as its genome. Two primary purposes of DNA  to make copies of itself so cells can divide and carry on the same information.  To carry instructions on how to make proteins so cells can build the machinery of life.
  12. 12. Structure of DNA  DNA is double helix.  DNA is composed of nucleotides  Nucleotides are made up of  Nucleobase(A,T,G,C)  Sugar  Phosphate
  13. 13. Base Pairing of DNA Strands A = T G C T = A A = T C G T C C A G G T A G C T = A T = A C G A = T A = T G C 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ denatured strands hybridized strands Hydrogen bonds C G C G G C Phosphate-sugar backbone ©2002 Academic Press 5’end | Phosphate | Sugar—Base… | Phosphate | Sugar—Base… | 3’end
  14. 14. Target Region for PCR chromosome cell nucleus Double stranded DNA molecule Individual nucleotides DNA in the Cell
  15. 15. Chromosomes • DNA found in the nucleus of the cell (nuclear DNA) is divided into chromosomes, which are dense packets of DNA and protection proteins called histones. The human genome consists of 22 matched pairs of autosomal chromosomes and two sex determining chromosomes. • Most human identity testing is performed using markers on the autosomal chromosomes and mitochondrial DNA while gender determination is done with markers on the sex chromosomes.
  16. 16. Some concepts….  Homologous: If the pair of chromosome has same size and same genetic material.  Heterologous: If the pair of chromosome do not have same size and genetic material.  Mitosis: Mitosis is the process of nuclear division in somatic cells that produces daughter cells, which are genetically identical to each other and to the parent cell.  Meiosis: Meiosis is the process of cell division in sex cells or gametes. In meiosis, two consecutive cell divisions result in four rather than two daughter cells, each with a haploid set of chromosomes.
  17. 17. cont….  Euchromatin: Regions of chromosomes that are transcriptionally active are known as euchromatin.  Heterochromatin: Regions of chromosomes that are transcriptionally inactive are known as heterochromatin.  Karyotype: The characterization of the chromosomal complement of an individual or a species, including number, form, and size of the chromosomes.
  18. 18. Chromosome Structure  Centromere: The center region of a chromosome, controls the movement of the chromosome during cell division.  On either side of the centromere are „arms‟ that terminate with telomeres. The shorter arm is referred to as „p‟ while the longer arm is designated „q‟. p (short arm) centromere telomere q (long arm) telomere Band 5 Band 3 Chromosome 12 12p3 12q5
  19. 19. Numbers of Chromosome reasoning  chromosomes are numbered based on their overall size with chromosome 1 being the largest and chromosome 22 the smallest. chromosome staining • A common method for staining chromosomes to obtain a banding pattern is the use of a Giemsa dye mixture that results in so-called ‘G-bands’ via the ‘G-staining’ method. These G-bands serve as signposts on the chromosome highway to help determine where a particular DNA sequence or gene is located compared to other DNA markers.
  20. 20. Genes  The coding regions are known as genes and contain the information necessary for a cell to make proteins. Humans have less than 30000 protein coding genes.  Two parts of a gene o Exons: coding region of gene o Introns: non-coding region of gene.  Only 5% of human genome are genes while 95% “junk DNA”.
  21. 21. Some concepts about Genes…  Locus: location of gene or marker on DNA in known as Locus.  Alleles: alternative possibilities for a gene are known as alleles.  Homozygous: if alleles are identical e.g. AA.  Heterozygous: if alleles are different e.g. Aa.  Genotype: A genotype is a characterization of the alleles present at a genetic locus.  DNA Profile: A DNA profile is the combination of genotypes obtained for multiple loci. DNA typing or DNA profiling is the process of determining the genotype present at specific locations along the DNA molecule.
  22. 22. Markers  A marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or species.  Markers used for human identity testing are found in the non-coding regions either between genes or within genes (i.e., introns).  The most recent and probably most rap- idly accepted forensic DNA markers are short tandem repeats (STRs).
  23. 23. ADVANTAGES OF STR MARKERS  They are PCR-based and work with low-quantity DNA templates or degraded DNA samples.  They are amenable to automation and involve sensitive fluorescent detection, which enables scientists to collect data quickly from these markers.  STRs are highly discriminating between unre- lated and even closely related individuals.
  24. 24. Nomenclature For DNA Markers  Two ways for naming a marker o If a marker is part of a gene or falls within a gene, the gene name is used in the designation. For example TH01. Sometimes the prefix HUM- is included at the beginning of a locus name to indicate that it is from the human genome. So HUMTH01. Here TH name of gene i.e human tyrosine hydroxylase. 01 The intron number of gene. i.e intron number 1 of gene.
  25. 25. Cont…… o If DNA markers fall outside of gene regions may be designated by their chromosomal position. For example D5S818. Here D DNA 5 Chromosome number i.e. 5 S Single stranded 818 The number of locus on chromosome
  26. 26. Product rule..  If each locus is inherited independent of the other loci, then a calculation of a DNA profile frequency can be made by multiplying each individual genotype frequency together. This is known as the product rule.
  27. 27. GENETIC VARIABILITY  With larger numbers of alleles for a particular DNA marker, a greater number of genotypes result. In general, if there are n alleles, there are n homozygous genotypes and n(n − 1)/2 heterozygous ones.  For example a locus with ten possible alleles would exhibit 10 homozygous possibilities plus [10×(10−1)]/2 heterozygous possibilities or 10 + 45 = 55 total genotypes.
  28. 28. Recombination  Recombination is the process by which progeny derive a combination of genes different from that of either parent.  In this manner, human genetic material is effectively shuffled with each generation producing the diversity seen in the world today.
  29. 29. GENBANK: A DATABASE OF DNA SEQUENCES  Genetic variation from DNA sequence information around the world is cataloged in a large computer database known as GenBank. GenBank is maintained by the National Center for Biotechnology Information (NCBI).  As of December 2003, GenBank contained over 36 billion nucleotide bases from more than 30 million different records.  This repository of DNA sequence information is not from humans alone. Over 120000 different species are represented in GenBank.
  30. 30. METHODS FOR MEASURING DNA VARIATION  Primary approaches for performing DNA typing  Restriction fragment length polymorphism (RFLP),  Polymerase chain reaction (PCR)-based methods.
  31. 31. Characteristic RFLP Methods PCR Methods Time required to obtain results 6-8 weeks with radioactive probes; ~1 week with chemiluminescent probes 1-2 days Amount of DNA needed 50-500 ng 0.1-1 ng Condition of DNA needed high molecular weight, intact DNA may be highly degraded Capable of handling sample mixtures Yes (single locus probes) Yes Allele identification Binning required Discrete alleles obtained Power of Discrimination ~1 in 1 billion with 6 loci ~1 in 1 billion with 8- 13 loci (requires more loci) Comparison of RFLP and PCR
  32. 32. Chapter 3 Sample Collection and Preparation (DNA Extraction and Quantification)
  33. 33. DNA Sample Sources  The most common materials in forensic laboratories are  Blood  Bloodstains  Semen  semen stains.
  34. 34. Blood and blood stains Budowle 1995 Semen and semen stains Budowle 1995 Bones Gill 1994 Teeth Alvarez 1996 Hair with root Higuchi 1988 Hair shaft Wilson 1995 Saliva (with nucleated cells) Sweet 1997 Urine Benecke 1996 Feces Hopwood 1996 Debris from fingernails Wiegand 1993 Muscle tissue Hochmeister 1998a Cigarette butts Hochmeister 1991 Postage stamps Hopkins 1994 Envelope sealing flaps Word 1997 Dandruff Herber 1998 Fingerprints Van Oorschot 1997 Personal Items: Razor blade, chewing gum, wrist watch, ear wax, toothbrush Tahir 1996 Material Reference Sources of Biological Evidence ©2002 Academic Press
  35. 35. Cont…  DNA evidence collection from a crime scene must be performed carefully and a chain of custody established in order to produce DNA profiles that are meaningful and legally accepted in court.  The evidence must be carefully collected, preserved, stored, and transported prior to any analysis conducted in a forensic DNA laboratory.  The National Institute of Justice has produced a brochure entitled „What Every Law Enforcement Officer Should Know About DNA Evidence‟ (see that contains helpful hints for law enforcement personnel who are the first to arrive at a crime scene.
  36. 36. Suggestions for Sample Collection  Avoid contaminating the area where DNA might be present by not touching it with your bare hands, or sneezing and coughing over the evidence.  Use clean latex gloves for collecting each item of evidence. Gloves should be changed between handling of different items of evidence.  Each item of evidence must be packaged separately.  Bloodstains, semen stains, and other types of stains must be thoroughly air-dried prior to sealing the package.  Samples should be packaged in paper envelopes or paper bags after drying. Plastic bags should be avoided because water condenses in them,  Stains on unmovable surfaces (such as a table or floor) may be transferred with sterile cotton swabs and distilled water.
  37. 37. COLLECTION OF REFERENCE DNA SAMPLES  In order to perform comparative DNA testing with evidence collected from a crime scene, biological samples must also be obtained from suspects or convicted felons.  Family reference samples are used in missing persons investigations, paternity testing, and mass disaster victim identifications.
  38. 38. PRESUMPTIVE TESTS  Prior to taking the effort to extract DNA from a sample, presumptive tests are often performed to indicate whether or not biological fluids such as blood or semen are present on an item of evidence (e.g., a pair of pants).  Three primary stains of forensic interest come from blood , semen, and saliva. Identification of vaginal secretions, urine, and feces can also be important to an investigation.  Serology is the term used to describe a broad range of laboratory tests that utilize antigen and serum antibody reactions. For example, the ABO blood group types are determined by serology.
  39. 39. DETECTION OF BLOOD STAINS  Blood is composed of liquid plasma and serum with solid components consisting of red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes).  Most presumptive tests for blood focus on detecting the presence of hemoglobin molecules, which are found in the red blood cells.  Two test are used for human blood testing  A simple immunochromatographic test  Luminol test
  40. 40. Cont…  A simple immunochromatographic test for identification of human blood is available from Abacus Diagnostics (West Hills, CA) as the ABAcard® HemaTrace® kit.  This test has a limit of detection of 0.07 μg hemoglobin/mL and shows specificity for human blood along with higher primate and ferret blood.  Luminol test can be used to locate traces of blood that have been diluted up to 10 million times.  The luminol reagent is prepared by mixing 0.1 g 3-amino-phthalhydrazide and 5.0 g sodium carbonate in 100 mL of distilled water. Before use, 0.7 g of sodium perborate is added to the solution (Saferstein 2001).
  41. 41. DETECTION OF SEMEN STAINS  Semen stains can be characterized with visualization of sperm cells, acid phosphatase (AP) or prostate specific antigen (PSA or p30) tests.  A microscopic examination to look for the presence of spermatozoa is performed in some laboratories on sexual assault evidence. But two issues exist…  Aspermic or oligospermic males have either no sperm or a low sperm count in their seminal fluid ejaculate.  Vasectomized males will not release sperm.
  42. 42. Acid phosphatase Detection  Acid phosphatase is an enzyme secreted by the prostate gland into seminal fluid and found in concentrations up to 400 times greater in semen than in other body fluids (Sensabaugh 1979, Saferstein 2001). A purple color with the addition of a few drops of sodium alpha naphthylphosphate and Fast Blue B solution or the fluorescence of 4-methyl umbelliferyl phosphate under a UV light indicates the presence of AP.  Prostate specific antigen was discovered in the 1970s and shown to have forensic value with the identity of a protein named p30 due to its apparent 30000 molecular weight (Sensabaugh 1978).
  43. 43. DIRECT OBSERVATION OF SPERM  A common method of doing this is to recover dried semen evidence from fabric or on human skin with a deionized water-moistened swab.  A portion of the recovered cells are then placed onto a microscope slide and fixed to the slide with heat. The immobilized cells are stained with a „Christmas Tree‟ stain.  „Christmas Tree‟ stain consists of aluminum sulfate, nuclear fast red, picric acid, and indigo carmine (Shaler 2002).  The stained slide is then examined under a light microscope for sperm cells with their characteristic head and long tail. The Christmas Tree stain marks the anterior sperm heads light red or pink, the posterior heads dark red, the spermatozoa‟s mid-piece blue, and the tails stain yellowish green (Shaler 2002).
  44. 44. DIRECT CAPTURE OF SPERM CELLS  Laser-capture microdissection, which is commonly used to select tumor cells from surrounding tissue on microscope slides. Sperm cells from sexual assault evidence spread on microscope slides can be collected with laser-capture microdissection to perform reliable STR testing (Elliot et al. 2003).  When sperm cells are observed in the field of view of the microscope, a tiny laser is activated and a thin plastic film placed over the slide melts at the specific point of laser light contact to capture the cell of interest. By moving the microscope slide around, dozens of sperm cells are collected onto this thin film that sits directly above the sample. The collection film is then transferred to a tube where DNA from the isolated sperm can be extracted and amplified using the polymerase chain reaction
  45. 45. DIRECT CAPTURE OF SPERM CELLS  Sperm cells can be collected on magnetic particles or beads that can be coated with antibodies specific for sperm proteins (Marshall 2002). The beads are then washed to remove the female epithelial cells. Finally, the purified sperm are placed into a PCR reaction to produce a DNA profile of the perpetrator.  This approach depends on sperm being intact, which is not always the case with old sexual assault evidence.
  46. 46. DETECTION OF SALIVA STAINS  A presumptive test for amylase is used for indicating the presence of saliva, which is especially difficult to see since saliva stains are nearly invisible to the naked eye  Saliva stains may be found on bite-marks, cigarette butts, and drinking vessels (Abaz et al. 2002, Shaler 2002).  Two common methods for estimating amylase levels in forensic samples include  Phadebas test  Starch iodine radial diffusion test
  47. 47. DNA EXTRACTION  A biological sample obtained from a crime scene a paternity case contains a number of substances besides DNA.  Cellular proteins that package and protect DNA in the environment of the cell can inhibit the ability to analyze the DNA. So, DNA molecules must be separated from other cellular material before they can be examined.  There are three DNA extraction methods  Organic extraction  Chelex extraction  FTA paper
  48. 48. Organic extraction or phenol chloroform extraction  Organic extraction used for situations where either RFLP or PCR typing is performed.  High molecular weight DNA, which is essential for RFLP methods, may be obtained most effectively with organic extraction.
  49. 49. Organic Extraction Procedure Blood stain SDS, DTT, EDTA and proteinase K Phenol, chloroform, isoamyl alcohol QUANTITATE DNA PERFORM PCR INCUBATE (56 oC) Centrifuge VORTEX Centrifuge CONCENTRATE sample (Centricon/Microcon-100 or ethanol precipitation) Centrifuge TRANSFER aqueous (upper) phase to new tube TE buffer
  50. 50. Chelex method  The Chelex method of DNA extraction is more rapid than the organic extraction method.  Chelex extraction involves fewer steps and thus fewer opportunities for sample-to-sample contamination.  However, it produces single stranded DNA as a result of the extraction process and therefore is only useful for PCR-based testing procedures.
  51. 51. Chelex Extraction Blood stain Water INCUBATE (ambient) 5% Chelex INCUBATE (100 oC) REMOVE supernatant INCUBATE (56 oC) QUANTITATE DNA PERFORM PCR Centrifuge Centrifuge
  52. 52. FTA Paper Extraction  FTA paper was developed by Lee Burgoyne at Flinders University in Australia as a method for storage of DNA (Burgoyne et al. 1994).  FTA paper is an absorbent cellulose-based paper that contains four chemical sub- stances to protect DNA molecules from nuclease degradation and preserve the paper from bacterial growth (Burgoyne 1996).  DNA on FTA paper is stable at room temperature over a period of several years.
  53. 53. Procedure  Add a spot of blood to the paper and allowing the stain to dry. The cells are lysed upon contact with the paper and DNA from the white blood cells is immobilized within the matrix of the paper.  A small punch of the paper is removed from the FTA card bloodstain and placed into a tube for washing. The bound DNA can then be purified by washing it with a solvent to remove heme and other inhibitors of the PCR reaction.  This purification of the paper punch can be seen visually because as the paper is washed, the red color is removed with the supernatant.  The clean punch is then added directly to the PCR reaction. Alternatively, some groups have performed a Chelex extraction on the FTA paper punch and used the supernatant in the PCR reaction (Lorente et al. 1998, Kline et al. 2002).
  54. 54. FTA Paper WASH Multiple Times with extraction buffer PCR Reagents Apply blood to paper and allow stain to dry PUNCH REMOVE supernatant PERFORM PCR (NO DNA QUANTITATION REQUIRED)
  55. 55. Storing DNA  Extracted DNA is typically stored at −20°C, or even −80°C for long-term stor- age, to prevent nuclease activity.  Nucleases are enzymes (proteins) found in cells that degrade DNA to allow for recycling of the nucleotide components.  Nucleases need magnesium to work properly so one of the measures to prevent them from digesting DNA in blood is the use of purple-topped tubes containing a blood preservative known as EDTA.  The EDTA chelates, or binds up, all of the free magnesium and thus prevents the nucleases from destroying the DNA in the collected blood sample.
  56. 56. DIFFERENTIAL EXTRACTION  Differential extraction is a modified version of the organic extraction method that separates epithelial and sperm cells.  Differential extraction was first described in 1985 (Gill et al. 1985).  Now commonly used by the FBI Laboratory and other forensic crime laboratories to isolate the female and male fractions in sexual assault cases that contain a mixture of male and female DNA.
  57. 57. Perpetrator‟s sperm mixed with victim‟s epithelial cells Centrifug e REMOVE supernatant SDS, EDTA and proteinase K (cell lysis buffer) Remove a portion of the mixed stain SDS, EDTA and proteinase K + DTT Incubate at 37 oC sperm pellet DTT lyses sperm heads “Male Fraction” “Female Fraction”sperm pellet
  58. 58. SOLID-PHASE EXTRACTION  Two approaches  QIAamp spin columns  DNA IQ™ system
  59. 59. QIAamp spin columns  In this approach nucleic acids selectively absorb to a silica support, such as small glass beads, in the presence of high concentrations of chaotropic salts such as guanidine hydrochloride, guanidine isothiocyanate, sodium iodide, and sodium perchlorate (Vogelstein and Gillespie 1979, Boom et al. 1990, Duncan et al. 2003).  These chaotropic salts disrupt hydrogen bonding networks in liquid water and thereby make denatured proteins and nucleic acids more thermodynamically stable than their correctly folded or structured counter- parts (Tereba et al. 2004).  If the solution pH is less than 7.5, DNA adsorption to the silica is typically around 95% and unwanted impurities can be washed away.  Under alkaline conditions and low salt concentrations, the DNA will efficiently elute from the silica material.
  60. 60. DNA IQ™ system  The DNA IQ™ system utilizes the same silica-based DNA binding and elution chemistries as QIAGEN kits but with silica-coated paramagnetic resin (Tereba et al. 2004).  With this approach, DNA isolation can be performed in a single tube by simply adding and removing solutions.  First, the DNA molecules are reversibly bound to the magnetic beads in solution with a solution pH of less than 7.5. A magnet is used to draw the silica- coated magnetic beads to the bottom or side of the tube leaving any impurities in solution. These solution impurities (proteins, cell debris, etc.,) can easily be removed by drawing the liquid off of the beads. The magnetic particles with DNA attached can be washed multiple times to more thoroughly clean the DNA. Finally, a defined amount of DNA can be released into solution via heating for a few minutes.
  61. 61. OTHER METHODS FOR DNA EXTRACTION  A simple closed tube DNA extraction method has been demonstrated with a thermal stable protease that looks very promising (Moss et al. 2003).  Microwave extraction has been used to shorten the conventional organic extraction method by several hours and to yield genomic DNA that could be PCR-amplified (Lee et al. 1994).  The addition of 6M NaCl to a proteinase K-digested cell extract followed by vigorous shaking and centrifugation results in a simple precipitation of the proteins (Miller et al. 1988). The supernatant containing the DNA portion of cell extract can then be added to a PCR reaction.  A simple alkaline lysis with 0.2M NaOH for five minutes at room temperature has been shown to work as well (Rudbeck and Dissing 1998, Klintschar and Neuhuber 2000).
  62. 62. PCR INHIBITORS AND DNA DEGRADATION  The presence of inhibitors or degraded DNA can manifest themselves by complete PCR amplification failure or a reduced sensitivity of detection usually for the larger PCR products.  Two PCR inhibitors commonly found in forensic cases are hemoglobin and indigo dyes from denim.  Melanin found in hair samples can be a source of PCR inhibition when trying to amplify mitochondrial DNA.  These inhibitors likely bind in the active site of the TaqDNA polymerase and prevent its proper functioning during PCR amplification.  Heat and humidity, which speed up hydrolytic cleavage, are enemies of intact DNA molecules. UV irradiation (e.g., direct sunlight) can lead to cross-linking of adjacent thymine nucleotides on the DNA molecule, which will prevent passage of the polymerase during PCR.
  63. 63. DNA QUANTITATION  Early methods for DNA quantitation typically involved either absorbance at a wavelength of 260nm or fluorescence after staining a yield gel with ethidium bromide.  Unfortunately, because these approaches are not very sensitive, they consume valuable forensic specimens that are irreplaceable. In addition, absorbance measurements are not specific for DNA and contaminating proteins or phenol left over from the extraction procedure can give falsely high signals.  Modern techniques are  Slot blot procedure  Fluorescence- based microtiter plate assays („real-time or quantitative PCR‟)
  64. 64. Slot Blot  Slot blot procedure test is specific for human and other primate DNA due to a 40 base pair (bp) probe that is complementary to a primate-specific alpha satellite DNA sequence D17Z1 located on chromosome 17 (Waye et al. 1989, Walsh et al. 1992).  The slot blot assay was first described with radioactive probes (Waye et al. 1989) but has since been modified and commercialized with chemiluminescent or colorimetric detection formats (Walsh et al. 1992).  Slot blots is fairly sensitive as it can detect both single-stranded and double- stranded DNA down to levels of approximately 150pg or about 50 copies of human genomic DNA. A 150pg DNA standard can be detected after only a 15 minutes exposure to X-ray film (Walsh et al. 1992).
  65. 65. Cont…  Slot blots involve the capture of genomic DNA on a nylon membrane followed by addition of a human-specific probe. Chemiluminescent or colorimetric signal intensities are compared between a set of standards and the samples.  Slot blot quantitation is a relative measurement involving the comparison of unknown samples to a set of standards that are prepared usually via a serial dilution from a DNA sample of known concentration.  While comparison of the digital capture and quantification of slot blot images has been demonstrated with a charged-coupled device (CCD) camera imaging system (Budowle et al. 2001). 20 ng 10 ng 5 ng 2.5 ng 1.25 ng 0.63 ng20 ng 10 ng 5 ng 2.5 ng 1.25 ng 0.63 ng Calibration standards Calibration standards Unknown Samples ~2.5 ng
  66. 66. PICOGREEN MICROTITER PLATE ASSAY  The Forensic Science Service has developed a PicoGreen assay that is capable of detecting as little as 0.25ng/mL of double-stranded DNA in a 96-well microtiter plate format (Hopwood et al. 1997).  PicoGreen is a fluorescent interchelating dye whose fluorescence is greatly enhanced when bound to double-stranded DNA.  To perform this microtiter plate assay, 5µL of sample are added to 195µL of a solution containing the PicoGreen dye. Each sample is placed into an individual well on a 96-well plate and then examined with a fluorometer. A 96-well plate containing 80 individual samples and 16 calibration samples can be analyzed in under 30 minutes (Hopwood et al. 1997).  Unfortunately, this assay quantifies total DNA in a sample and is not specific for human DNA.
  67. 67. ALUQUANT™ HUMAN DNA QUANTITATION SYSTEM  The Promega Corporation has developed a human DNA quantitation system that enables sensitive detection of DNA (Mandrekar et al. 2001). The AluQuant™ assay probes Alu repeats that are in high abundance in the human genome.  Probe-target hybridization initiates a series of enzymatic reactions that end in oxidation of luciferin with production of light. The light intensity is read by a luminometer and is proportional to the amount of DNA present in the sample.  The AluQuant™ assay possesses a range of 0.1–50ng for human DNA and can be automated on a robotic liquid handling workstation (Hayn et al. 2004)
  68. 68. END-POINT PCR FOR DNA QUANTIFICATION  A approach for testing the „amplifiability‟ of a DNA sample is to perform an end-point PCR test. In this approach a single STR locus (Kihlgren et al. 1998, Fox et al. 2003) or other region of the human genome, such as an Alu repeat (Sifis et al. 2002, Nicklas and Buel 2003a), is amplified along with DNA samples of known concentrations.  A standard curve can be generated from the samples with known amounts to which samples of unknown concentration are compared. A fluorescent intercalating dye such as SYBR® Green can be used to detect the generated PCR products.  Based on the signal intensities resulting from amplification of the single STR marker or Alu repeat region, the level of DNA can be adjusted prior to amplifying the multiplex set of DNA markers in order to obtain the optimal results.
  69. 69. Calculation of the quantity of DNA in a cell 1. Molecular Weight of a DNA Basepair = 618g/mol A =: 313 g/mol; T: 304 g/mol; A-T base pairs = 617 g/mol G = 329 g/mol; C: 289 g/mol; G-C base pairs = 618 g/mol 2. Molecular weight of DNA = 1.85 x1012 g/mol There are 3 billion base pairs in a haploid cell ~3 x 109 bp (~3 x 109 bp) x (618 g/mol/bp) = 1.85 x 1012 g/mol 3. Quantity of DNA in a haploid cell = 3 picograms 1 mole = 6.02 x 1023 molecules (1.85 x 1012 g/mol) x (1 mole/6.02 x 1023 molecules) = 3.08 x 10-12 g = 3.08 picograms (pg) A diploid human cell contains ~6 pg genomic DNA 4. One ng of DNA contains the DNA from 167 diploid cells 1 ng genomic DNA (1000 pg)/6pg/cell = ~333 copies of each locus (2 per 167 diploid genomes)
  70. 70. Chapter 4 The Polymerase Chain Reaction (DNA Amplification)
  71. 71. PCR Process  First described in 1985 by Kary Mullis and members of the Human Genetics group at the Cetus Corporation (now Roche Molecular Systems), PCR has revolutionized molecular biology with the ability to make millions of copies of a specific sequence of DNA in a matter of only a few hours.  This molecular process involves heating and cooling samples in a precise thermal cycling pattern over ~30 cycles. 94 oC 60 oC 72 oC Time Temperature Single Cycle Typically 25-35 cycles performed during PCR 94 oC 94 oC 94 oC 60 oC60 oC 72 oC72 oC The denaturation time in the first cycle is lengthened to ~10 minutes when using AmpliTaq Gold to perform a “hot-start” PCR
  72. 72. Separate strands (denature) Add primers (anneal) Make copies (extend primers) Repeat Cycle, Copying DNA Exponentially Starting DNA Template 5’ 5’ 3’ 3’ 5’ 5’ 5’ 3’ 3’ 3’3’5’ Forward primer Reverse primer
  73. 73. PCR  PCR is commonly performed with a sample volume in the range of 5–100µL. At such low volumes, evaporation can be a problem and accurate pipetting of the reaction components can become a challenge.  On the other hand, larger solution volumes lead to thermal equilibrium issues for the reaction mixture because it takes longer for an external temperature change to be transmitted to the center of a larger solution than a smaller one.  Therefore, longer hold times are needed at each temperature, which leads to longer overall thermal cycling times. Most molecular biology protocols for PCR are thus in the 20–50µL range.
  74. 74. PCR COMPONENTS  The most important components of a PCR reaction are the two primers, which are short DNA sequences that precede or „flank‟ the region to be copied.  The other components of a PCR reaction consist of  Template DNA that will be copied.  Building blocks made up of each of the four nucleotides  DNA polymerase that adds the building blocks in the proper order based on the template DNA sequence.
  75. 75. CONTROLS USED TO MONITOR PCR  A „negative control‟, which is the entire PCR reaction mixture without any DNA template. The negative control usually contains water or buffer of the same volume as the DNA template, and is useful to assess whether or not any of the PCR components have been contaminated by DNA (e.g., someone else in your lab).  A „positive control‟ is a valuable indicator of whether or not any of the PCR components have failed or were not added during the reaction setup phase of experiments conducted. The DNA template should be amplified with the same PCR primers as used on the rest of the samples in the batch that is being amplified. The purpose of a positive control is to ensure confidence that the reaction components and thermal cycling parameters are working for amplifying a specific region of DNA.
  76. 76. Whole Genome Amplification (WGA)  WGA involves a different DNA polymerase than the TaqGold enzyme commonly used in forensic DNA analysis. WGA amplifies the entire genome using random hexamers as priming points.  The WGA enzymes work by multiple displacement amplification (MDA), which is sometimes referred to as rolling circle amplification. MDA is isothermal with an incubation temperature of 30°C and requires no heating and cooling like PCR.
  77. 77. THERMAL CYCLERS  The instrument that heats and cools a DNA sample in order to perform the PCR reaction is known as a thermal cycler. Precise and accurate sample heating and cooling is crucial to PCR in order to guarantee consistent results.  The most prevalent thermal cycler in forensic DNA laboratories is the GeneAmp® PCR System 9600 from Applied Biosystems (Foster City, CA). The „9600‟ can heat and cool 96 samples in an 8 ×12-well microplate format at a rate of approximately 1°C per second.
  78. 78. Hot Start PCR  At a low temperature for the primers to bind to each other creating products called „primer dimers.‟ These are a particular problem because their small size relative to the PCR products means that they will be preferentially amplified.  Low-temperature mispriming can be avoided by initiating PCR at an elevated temperature, a process usually referred to as „hot start‟ PCR. Hot start PCR may be performed by introducing a critical reaction component, such as the polymerase, after the temperature of the sample has been raised above the desired annealing temperature (e.g., 60°C).  A more important disadvantage is the fact that the sample tubes must be opened at the thermal cycler to introduce the essential component, which gives rise to a greater opportunity for cross-contamination between samples.
  79. 79. AMPLITAQ GOLD DNA POLYMERASE  AmpliTaq Gold™ DNA polymerase is a chemically modified enzyme that is rendered inactive until heated (Birch et al. 1996). An extended pre- incubation of 95°C, usually for 10 or 11 minutes, is used to activate the AmpliTaq Gold.  AmpliTaq Gold is not compatible with the pH 9.0 buffers used for regular AmpliTaq DNA polymerases (Moretti et al. 1998). This fact is because the pH does not get low enough to remove the chemical modifications on TaqGold and thus the enzyme remains inactive. Tris buffers with a pH 8.0 or 8.3 at 25°C work the best with TaqGold.
  80. 80. MULTIPLEX PCR  The PCR permits more than one region to be copied simultaneously by simply adding more than one primer set to the reaction mixture (Edwards and Gibbs 1994). The simultaneous amplification of two or more regions of DNA is commonly known as multiplexing or multiplex PCR.  For a multiplex reaction to work properly the primer pairs need to be compatible. The primer annealing temperatures should be similar and excessive regions of complementarity should be avoided to prevent the formation of primer-dimers that will cause the primers to bind to one another instead of the template DNA.
  81. 81. REAL-TIME (QUANTITATIVE) PCR  Real-time PCR, which was first described by Higuchi and co-workers at the Cetus Corporation in the early 1990s (Higuchi et al. 1992, Higuchi et al. 1993), is sometimes referred to as quantitative PCR or „kinetic analysis‟ because it analyzes the cycle-to-cycle change in fluorescence signal resulting from amplification of a target sequence during PCR.  This analysis is performed without opening the PCR tube and therefore can be referred as a closed-tube or homogeneous detection assay.  The most common approaches utilize either the fluorogenic 5′ nuclease assay better known as TaqMan®.
  82. 82. THE 5′ NUCLEASE ASSAY (TAQMAN)  TaqMan probes are labeled with two fluorescent dyes that emit at different wavelengths. The probe sequence is intended to hybridize specifically in the DNA target region of interest between the two PCR primers (Ong and Irvine 2002). Typically the probe is designed to have a slightly higher annealing temperature compared to the PCR primers so that the probe will be hybridized when extension (polymerization) of the primers begins  The „reporter‟ (R) dye is attached at the 5′-end of the probe sequence while the „quencher‟ (Q) dye is synthesized on the 3′-end. A popular combination of dyes is FAM or VIC for the reporter dye and TAMRA for the quencher dye.  When the probe is intact and the reporter dye is in close proximity to the quencher dye, little-to-no fluorescence will result because of suppression of the reporter fluorescence due to an energy transfer between the two dyes.
  83. 83. Cont…  The Taq DNA polymerase used has a 5′-exonuclease activity and therefore will begin to chew away at any sequences in its path (i.e., those probes that have annealed to the target sequence). When the reporter dye molecule is released from the probe and it is no longer in close proximity to the quencher dye, it can begin to fluoresce.
  84. 84. Polymerization and Strand Displacement R Q Forward primer Reverse primer 3‟ 5‟ 3‟ 5‟ 3‟5‟ 5‟ 5‟ Forward primer Reverse primer 3‟ 5‟ 3‟ 5‟ 5‟ 5‟ Q R 3‟ Probe Cleavage (release of reporter dye) Forward primer Reverse primer 3‟ 5‟ 3‟ 5‟ 5‟ 5‟ Q Completion of Polymerization TaqMan probe Fluorescence occurs when reporter dye and quencher dye are no longer in close proximity
  85. 85. ADVANTAGES OF PCR WITH FORENSIC SPECIMENS  Very small amounts of DNA template may be used from as little as a single cell.  DNA degraded to fragments only a few hundred base pairs in length can serve as effective templates for amplification.  Large numbers of copies of specific DNA sequences can be amplified simultaneously with multiplex PCR reactions.  Contaminant DNA, such as fungal and bacterial sources, will not amplify because human-specific primers are used.  Commercial kits are now available for easy PCR reaction setup and amplification.
  86. 86. DISADVANTAGES OF PCR WITH FORENSIC SPECIMENS  The target DNA template may not amplify due to the presence of PCR inhibitors in the extracted DNA .  Amplification may fail due to sequence changes in the primer-binding region of the genomic DNA template.  Contamination from other human DNA sources besides the forensic evidence at hand or previously amplified DNA samples is possible without careful laboratory technique and validated protocols.