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DNA

DNA. Chapter 13. What is DNA?. DNA Structure. DNA stands for deoxyribonucleic acid Long molecule made up of nucleotides Consists of a 5- carbon sugar, phosphate group, and a nitrogenous base (adenine, guanine, cytosine, and thymine) Backbone of DNA is sugar and phosphate

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DNA

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  1. DNA Chapter 13

  2. What is DNA?

  3. DNA Structure • DNA stands for deoxyribonucleic acid • Long molecule made up of nucleotides • Consists of a 5- carbon sugar, phosphate group, and a nitrogenous base (adenine, guanine, cytosine, and thymine) • Backbone of DNA is sugar and phosphate • Nitrogenous bases stick out SUGAR Phosphate SUGAR Phosphate SUGAR Phosphate SUGAR A G C T

  4. DNA • Is found in all nucleated body cells • White blood cells • Semen • Saliva • Urine • Hair • Root • Teeth • Bone • Tissue • Is most abundant in buccal (cheek) cells

  5. Chromosomes • Composed of tightly coiled DNA and proteins • Humans have 46 chromosomes • Only visible during cell division • Consists of 2 arms called chromatids held together by the centromere

  6. Genes • Are portions of DNA that code for specific proteins • Is the fundamental unit of heredity • Each gene(s) codes for specific traits • Eye color, hair color, height, etc

  7. Historical Information

  8. James Watson and Francis Crick • Determined that DNA is a double helix

  9. Historical Milestones • 1988 • FBI starts DNA casework • 1991 • First STR (short tandem repeat) paper • 1998 • FBI launches CODIS database

  10. Base Pairing, Replication, and the Genetic Code

  11. Complementary Base Pairing • Is based on Chargaff’s rules • Adenine pairs with Thymine • Cytosine pairs with Guanine • Ex:) ATTGCGTAGGGCTA (TEMPLATE) TAACGCATCCCGAT (COMPLEMENT)

  12. DNA Replication • Before a cell divides, it duplicates its DNA • Process called replication • Process involves the DNA molecule separating into 2 strands and then producing two new complementary strands • Each strand of the DNA acts as a “template” • Occurs until each chromosome is copied

  13. DNA Replication • Is carried out by enzymes that “unzip” the DNA molecule • Occurs when hydrogen bonds between the base pairs are broken • Principle enzyme involved is DNA polymerase • Polymerizes nucleotides to produce DNA • Also proofreads each DNA strand

  14. RNA • Is transcribed from DNA through the process of transcription • A goes with U instead of T • G goes with C • After transcription comes translation • Pairing up of codons with anticodons on tRNA to form an amino acid

  15. Genetic Code • Proteins made by joining amino acids into chains called polypeptides • There are twenty amino acids • Properties of proteins determined by the order in which these amino acids are in • Language of mRNA instructions called genetic code

  16. Genetic Code • RNA has four bases- A, U, G, C • Genetic code read in threes • Called a codon • Each three letters represents a specific amino acid • Can be specified by more than one codon

  17. DNA Typing

  18. DNA Typing • Is a method in which DNA is converted into a series of bands that ultimately distinguish each individual • Involves repeating segments of DNA numerous times • Called tandem repeats • Act as fillers or spaces between the coding regions of DNA • Only 1/10th of a single percent of DNA differs from one person to the next • Used to generate DNA profiles of an individual

  19. DNA Typing • Has coding and non-coding regions • 3% of the human DNA sequences code for proteins • 97% is non-coding and is repetitive • Same sequences repeated over and over • Are the tandem repeats

  20. DNA Typing • 3 processes utilized • RFLP • Restriction Fragment Length Polymorphism • PCR • Polymerase Chain Reaction • STR • Short Tandem Repeats

  21. RFLP • Restriction enzymes are used to cut DNA into smaller fragments that can be separated and characterized for identification • Steps involved • Isolate • Separate DNA from the cell • Cut • Using restriction enzymes to make shorter base strands • Sort • By size using electrophoresis • Analyze • The specific alleles for identification

  22. RFLP • To visualize the RFLP’s, the fragments are transferred to a nylon membrane that has been treated with radioactive probes containing a base sequence complementary to the RFLP’s being identified • The nylon sheet is then placed against X-ray film and is exposed for several days • Bands appear where radioactive probes stuck to fragments on the nylon sheet • Usually 2 bands will show up (one RFLP from each chromosome) • Bands are analyzed to see similarities and/or differences

  23. PCR • Is a technique used for making copies of a defined segment of a DNA molecule • Can be valuable when the amount of evidence is minimal • Millions of copies of DNA can be made from a single speck of blood • DNA taken from white blood cells, not red blood cells

  24. PCR • Steps involved • Heat the DNA strands • Causes the strands to separate • Cool the mixture and add a primer • Is a short sequence of base pairs that will add to its complementary sequences on the DNA strand • Add DNA polymerase and a mixture of free nucleotides to the separated strands • Heat again to around 75 degrees Celsius for the completion

  25. PCR • The outcome of one cycle of PCR is a doubling of the number of DNA strands • Usually 25-30 cycles of PCR occur • Yield more than 1 million copies of the original DNA molecule • Each cycle takes 2 minutes to complete

  26. PCR • Advantages • Minute amounts of DNA may be used for amplification • 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

  27. PCR • Disadvantages • Contaminant DNA, such as those contaminated with fungus or bacteria, will not amplify

  28. Electrophoresis • Is a technique used to separate DNA fragments • An electrical current is moved through a gel substance causing molecules to sort by size • The smaller, lighter molecules will move the furthest on the gel • After developing, the fragments can be visualized for characterization

  29. Electrophoresis • Steps • Pipette the DNA • Load DNA into the gel wells • Run the gel • Observe and compare bands of DNA

  30. Short Tandem Repeats • AKA STR • Are locations on the chromosome that contain short sequences of 2-5 bases that repeat themselves in the DNA molecule • Entire strand of DNA is less than 450 bases in length • Is the most used procedure for DNA typing • Advantages • Provides greater discrimination • Requires less time • Requires smaller sample size • DNA is less susceptible to degradation

  31. Short Tandem Repeats • Procedure • Extract the gene TH01 from the sample • TH01 has seven human variants with a repeating sequence of AATG • Amplify the sample by means of PCR • Separate by electrophoresis • Examine the distance the STR migrates to determine the number of times TH01 repeats

  32. Short Tandem Repeats • Each person has two STR types for TH01 • One inherited from each parent • By continuing the process with additional STR’s from other genes, you can narrow down the probability of DNA belonging to only one person

  33. Short Tandem Repeats • 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 location (loci)

  34. Short Tandem Repeats • http://www.dnai.org/d/index.html

  35. Y-STR • Is another tool available to type STR’s located on the Y chromosome • Is male specific • More than 20 different Y-STR markers have been identified • Useful when multiple males are involved in a sexual assault • Analysis will have only one band or peak • Is less complicated in appearance and interpretation

  36. Outcomes to DNA Typing • 3 outcomes • Match • DNA profile appears the same • Lab determines the frequency • Exclusion • The genotype comparison shows profile differences that can only be explained by the two samples originating from different sources • Inconclusive • The data does not support a conclusion as to whether the profiles match

  37. Nuclear Found in the nucleus Constitutes 23 pairs of chromosomes inherited from both parents Each cell contains only one nuclei Mitochondrial Found in the cytoplasm Is inherited only from the mother Each cell contains hundreds to thousands of mitochondria Can be found in skeletal remains Types of DNA

  38. Mitochondrial DNA • Analysis of mDNA is more • Rigorous • Time consuming • Costly than nucleic testing of DNA • mDNA is constructed in a circle or loop • 37 genes are involved in mitochondrial energy generation • Is used when nuclear DNA typing is not possible

  39. CODIS • Stands for Combined DNA Index System • Used for linking serial crimes and unresolved cases with repeat offenders • Launched October 1998 • Links all 50 states • Locally, statewide, nationally • Requires >4 RFLP markers and/or 13 core STR markers

  40. Packaging Biological Evidence • Before packaging, photograph and record evidence on sketches • Wearing disposable latex gloves while handling the evidence is required • Clothing from victim and suspect with blood evidence must be collected • The packaging of biological evidence in plastic or airtight containers must be avoided • Moisture can contribute to growth of DNA-destroying bacteria and fungi

  41. Packaging Biological Evidence • Each stained article should be packaged separately in a paper bag or in a well- ventilated box • Dried blood is best removed using a sterile cotton swab lightly moistened with distilled water • Is air dried before being placed in a swab box, then a paper or manilla envelope • All biological evidence should be refrigerated or stored in a cool location until delivery to the lab • Standard/reference DNA specimens must also be collected

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