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DNA Fingerprinting

DNA Fingerprinting. Historical Information. James Watson and Francis Crick--1953 discovered the configuration of the DNA molecule Ray White--1980 describes first polymorphic RFLP marker Alec Jeffreys--1985 isolated DNA markers and called them DNA fingerprints

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DNA Fingerprinting

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  1. DNA Fingerprinting

  2. Historical Information • James Watson and Francis Crick--1953 discovered the configuration of the DNA molecule • Ray White--1980 describes first polymorphic RFLP marker • Alec Jeffreys--1985 isolated DNA markers and called them DNA fingerprints • Kary Mullis--1985 developed PCR testing • 1988--FBI starts DNA casework • 1991--first STR paper • 1998--FBI launches CODIS database.

  3. DNA Body Locations • Can be found in all body cells--blood, semen, saliva, urine, hair, teeth, bone, tissue • Most abundant in our buccal (cheek) cells • Blood is 99.9% red blood cells that have no nuclei; and therefore, no nuclear DNA • DNA obtained from blood comes from white blood cells

  4. DNA TYPING DNA typing is a method in which DNA is converted into a series of bands that ultimately distinguishes each individual. Only one-tenth of a single percent of DNA (about 3 million bases) differs from one person to the next. Scientists use these regions to generate a DNA profile of an individual.

  5. Non-Coding Regions • 3 percent of the human DNA sequences code for proteins • 97 percent is non-coding and is repetitive; repeating the same sequence over and over • 50 percent of the human genome has interspersed repetitive sequences

  6. General DNA Typing Process • Obtain a DNA sample. • Amplify DNA sample if needed • Cut the DNA into small pieces at certain chromosomes landmarks where there is variation from person to person. • Load and run electrophoresis. This separates the pieces by length. • Visualize the bands.

  7. DNA TYPING“Fingerprinting” • **Amplify DNA by PCR First • RFLP-- Restriction Fragment Length Polymorphism • STR--Short Tandem Repeats • Mitochondrial--use of maternal DNA in the mitochondria

  8. Advantages of PCR • Minute 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. • Commercial kits are now available for easy PCR reaction setup and amplification. Problem: Contaminant DNA, such as fungal and bacterial sources, will not amplify because human-specific primers are used.

  9. RFLP--Restriction Fragment Length Polymorphisms • Fragments are cut from the sequence of bases by a restriction enzyme. • The enzyme find its combination, bonds at one end and dissolves through the DNA at the other. • Fragments are loaded into a gel and run by electrophoresis. • DNA is extracted from the gel by blotting it into a nylon membrane.

  10. RFLP--Restriction Fragment Length Polymorphisms • Radioactive phosphorus-32 probes are added to the membrane which bond to the precise DNA fragments making them radioactive. • Then the membrane is placed over standard X-ray film where the radiation emitted from the P-32 gradually exposes the film and shows the DNA bands. This process takes about 10 weeks to complete.

  11. Short Tandem Repeats (STR) STR is the latest method of DNA typing. STR’s are locations (loci) on the chromosome that contain short sequences of 3 to 7 bases that repeat themselves with the DNA molecule. This method’s advantages include a higher discrimination than RFLP, less time, smaller sample size, and less susceptible to degradation.

  12. Short Tandem Repeats STRsCounting the number of repeats by size comparison Largest AGCT AGCT AGCT AGCT 4 Repeats AGCT AGCT AGCT 3 Repeats AGCT AGCT 2 Repeats AGCT 1 Repeat Smallest

  13. Short Tandem Repeats (STR) • Extract the gene TH01 from the sample. (TH01 has seven human variants and a repeating sequence of A-A-T-G) • Amplify the sample by means of PCR • Separate by electrophoresis • Examine the distance the STR migrates to determine the number of times TH01 repeats

  14. Short Tandem Repeats (STR) Each person has two STR types for TH01--one inherited from each parent. By continuing the process with additional STRs from other genes, you can narrow down the probability of DNA belonging to only one possible person.

  15. STR 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 loci.

  16. Profiler Plus Allelic Ladders VWA D3S1358 FGA AMEL D8S1179 D21S11 D18S51 D13S317 D5S818 D7S820

  17. COfiler Allelic Ladders D3S1358 D16S539 AMEL TH01 TPOX CSF1PO D7S820

  18. My STR’s

  19. Probability of Matching My STR’s

  20. Three Possible Outcomes • Match--The DNA profile appears the same. Lab will determine 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.

  21. Nuclear found in the nucleus constitutes 23 pair of chromosomes inherited from both parents each cell contains only one nuclei Mitochondrial found in the cytoplasm is inherited only from mother each cell contains hundreds to thousands of mitochondria can be found in skeletal remains Types of DNA Nuclear DNA is present in the head of the sperm. Mitochondrial DNA is present in the tail. At conception, the head of the sperm enters the egg and unites with the nucleus. The tail falls off, losing the father’s mitochondrial DNA.

  22. Mitochondrial DNA • Analysis of mtDNA is more: • rigorous • time consuming • costly than nucleic testing of DNA • mtDNA is constructed in a circular or loop • 37 genes are involved in mitochondrial energy generation • Is best used when nuclear DNA typing is not possible

  23. Determining Probability Databases are established by which one has determined how often a particular allele on a loci appears in a given population. By increasing the number of alleles on different loci the probability of having two people with the exact combination becomes astronomical.

  24. Probability of a Random Match Using 13 CODIS STR Markers

  25. Probability Numbers:What do they mean? 1,000,000 million 1,000,000,000 billion 1,000,000,000,000 trillion 1 x 1015quadrillion 1 x 1018quintillion 1 x 1021 sextillion 1 x 1024septillion 1 x 1027octillion 1 x 1030nonillion 1 x 1033decillion

  26. Present Work in DNA Fingerprinting • Forensics • Missing Persons • Paternity/Maternity • Genetics

  27. The Future • Greater automation of the DNA typing process • Use of SNP’s--single nucleotide polymorphism which measures a one nucleotide change or difference from one individual to another. More sites are needed to differentiate between individuals (30 to 50 SNPs to attain the frequencies of the 13 STR loci), but it can be done with robots and automation.

  28. Problems? • Ethics involved in DNA Fingerprinting for health • Insurance companies • Job Screening • Lethal Diseases • Legal issues arising from genome sequencing • Use of “I’m genetically predisposed”

  29. WORDS OF WISDOM Keep in mind a famous quote from Albert Einstein, “ If we knew what we were doing, it would not be called research, would it?”

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