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DNA

DNA. DNA. Deoxyribonucleic acid Instructions for life Nucleus Double helix Arranged in chromosomes. Gene . * Basic unit of heredity * Segment of a chromosome that codes for a protein. Humans have 46 chromosomes 23 pairs in every cell except for: A. RBCs B. eggs and sperm (1/2)

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DNA

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  1. DNA

  2. DNA • Deoxyribonucleic acid • Instructions for life • Nucleus • Double helix • Arranged in chromosomes

  3. Gene * Basic unit of heredity * Segment of a chromosome that codes for a protein

  4. Humans have 46 chromosomes • 23 pairs in every cell except for: • A. RBCs • B. eggs and sperm (1/2) • 8 trillion possible combinations from just 2 parents

  5. DNA • Made of nucleotides • Sugar, phosphate, base • Double helix

  6. Bases • A, G, C, T • Purines • Pyrimidines • Rule of base pairing • 3 billion base pairs

  7. Sources of DNA • Skin • Hair follicles • Semen • Saliva • Blood

  8. Genome Total DNA within a cell 5% -- encoded genes 95% - non-encoded (junk) may regulate genes most of it … no known purpose

  9. Genome • Over 99% of your genome is the same as everyone else’s. • Alec Jeffrey’s :1985 found that each person’s DNA is unique (basis of DNA fingerprinting= DNA typing)

  10. Preserving DNA • Gloves • Dried or frozen • Need big enough fragments to analyze • 20 years ago – needed 25-100X more DNA

  11. DNA fingerprinting Humans – 3 billion base pairs - too much to cut w/ enzymes and run all of it thru electrophoresis -too many fragments … bands smeared Good news!! – differences in DNA between people are found in certain areas on the chromosomes. --tandem repeats

  12. Analyzing DNA • RFLP • PCR • VNTR • STR • mtDNA

  13. RFLP = Restriction fragment length polymorphism • PCR = Polymerase chain reaction • VNTR = variable number tandem repeats

  14. STR = Short tandem repeat mtDNA = mitochondrial DNA

  15. RFLP • Analyzes variable lengths of DNA fragments • Fragments made by cutting with restriction enzymes (endonucleases) • Cuts DNA at a specific sequence know as a recognition site. • Presence/absence of certain recognition sites produces fragments of variable lengths

  16. Fragments separated using gel electrophoresis. • Fragments are hybridized with DNA probes that bind to a complementary DNA sequence in the sample.

  17. DNA probes • Nucleotides that are radioactive or that fluoresce are used to make the probes. • Probes are then ”visible” • Probes are short sequences that bind to a specific area of the DNA

  18. PCR Used to make copies of DNA (amplify) Primers- short single-stranded pieces of DNA…. Complementary to the start of the fragment that you want to amplify

  19. PCR In tube: Target DNA Primers DNA polymerase Buffer A,T,G,C

  20. Cycles • Heat - breaks hydrogen bonds of double helix • Cool- primers bind to target DNA • DNA polymerase adds new nucleotides to the primer • DNA is copied • Repeat many times

  21. PCR • Total DNA = 2n • Ex. 30 cycles • 230 times the original DNA = more than a billion new DNA fragments

  22. PCR websites • http://allserv.rug.ac.be/~avierstr/principles/pcr.html

  23. Some areas of DNA show polymorphism. • Polymorphisms – “many forms” • Found in non-encoded DNA (junk) • Variable areas in DNA • Variable in length and base sequence • Ex. Length polymorphism

  24. Some base sequences in the junk DNA are constantly repeated. VNTRs STRs

  25. Tandem repeats • End-to end repeats of different short DNA sequences • 3 -30+ bases at many places scattered thru chromosomes.

  26. Tandem repeats • Locations of repeats are same from person to person • Base sequences of repeats are same from person to person • # of repeats varies from person to person

  27. Therefore, the DNA fragments that result from those areas are different sizes • Need to look at results from many regions of the chromosome.

  28. VNTR = variable number tandem repeats Repeats of the same base sequence May be hundreds of base pairs long Will repeat a variable # of times

  29. STR = short tandem repeats 3-7 base pairs long Will repeat a variable # of times May extend over a distance of 400 bases Can use severely degraded DNA

  30. Variation Researchers have determined how often a given # of repeats is found at a certain locus. Can be used to calculate probability.

  31. Location # 1 = 1/20 chance (of matching someone else) Location # 2 = 1/10 chance Location # 3 = 1/5 chance Total = 1 x 1 x 1 = 1 20 10 5 1000

  32. Procedure • Extract the DNA • Cutting or amplifying DNA • Separate fragments w/ electrophoresis • Transfer fragments to a nylon membrane • Tag fragments w/ DNA probe • Visualize fragments thru x-ray

  33. Making the Match • Bands match in known and unknown samples. • Lanes control (to estimate size of fragments) crime-scene victim suspect

  34. CODIS • Combined DNA Index System • Database • Local, state, national system • LDIS, SDIS,NDIS • Free set-up, training • Legal to collect DNA samples from convicted offenders

  35. CODIS Forensic index: DNA from crime scene Offender Index: convicted felons

  36. HITS • 9/99 – 600 hits • 9/03 – 9000 hits • 9/04 – 17,200 hits

  37. Case study Green River Killer 50 prostitutes Seattle 82-91 semen sample too small got saliva from main suspect 1987 mid 90’s – PCR, STR used to connect saliva/sperm

  38. http://science.howstuffworks.com/dna-evidence3.htm http://www.genelex.com/paternitytesting/paternitybook.html http://www.wiley.com/legacy/college/boyer/0470003790/animations/animations.htm http://www.pbs.org/wgbh/nova/sheppard/analyze.html

  39. http://www.scienceteacherprogram.org/biology/simon02.html

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