University:
Boston University
Course:
CAS BI 107 | Biology 1
Academic year:

2024
Views:

443

Pages:

15
Author:

Edward Ritter

Overview • Background ~30 min – DNA profiling – Polymerase Chain Reaction (PCR) – Gel Electrophoresis • Lab activity ~50 min – PCR What is DNA profiling • The use of molecular genetic methods to determine the exact genotype of a DNA sample in a way can distinguish one human being from another. How do crime scene investigators create a DNA profile? • Step #1 Evidence is collected at the crime scene • Biological sources: anything that has DNA! Blood Tissue Semen Urine Hair Teeth Saliva Bone Step #2 DNA is Extracted Chromosome • Sources at scene • Victim • Suspects Cell DNA Step #3 DNA samples are processed Sample Obtained from Crime Scene or Paternity Investigation DNA Extraction Biology DNA Quantification PCR Amplification of Multiple STR markers Technology Separation and Detection of PCR Products (STR Alleles) 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 How are suspects included or excluded from an investigation? • Suspects are included in an investigation if their DNA profile matches with genotypes found at the crime scene • Suspects can be excluded if their DNA profile does not match genotypes found at the crime scene What part of DNA is used? • There are ~3 billion base pairs in the human genome • >99.5% of human DNA sequences are the same in every person • Small percentage of DNA is different allowing us to distinguish one individual from another • Polymorphic DNA – “many forms” • Do not control any traits, and have no function Variable Number Tandem Repeat (VNTR) • DNA profiling uses repetitive ("repeat") sequences that vary a lot • VNTR loci are very similar between closely related humans. (relatives) • However, they are so variable that unrelated individuals are extremely unlikely to have the same VNTRs. • Amplify loci and visualize to see differences Short Tandem Repeats (STR’s) • Type of VNTR – Variable number tandem repeat • Unique from individual to individual and are “anonymous” • Control no known trait or function STR region Example of an STR • TH01 locus: Tyrosine hydroxylase intron 01 • Repeats of TCAT • CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA • This example has 6 TCAT repeats. • There are more than 20 known TH01 alleles. • Each individual inherits 1 allele from each parent. • These make up the genotype. Ex. 6,2 Example of an STR • Ms. Smith’s TH01 locus for her two chromosomes is given below. • What is her genotype? • MOM’S CHROMOSOME CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA • DAD’S CHROMOSOME CCC TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT AAA Example of an STR • Ms. Smith’s TH01 locus for her two chromosomes is given below. • MOM’S CHROMOSOME – CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA • DAD’S CHROMOSOME – CCC TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT AAA • What is her genotype? • How can we visualize this difference? Polymerase Chain Reaction (PCR) • • • • Replication in a test tube Makes copies of a specific piece of DNA Amplify DNA so we can visualize it Allows you to look at one specific piece of DNA by making copies of *only* that piece of DNA • Can amplify a very small amount of DNA • PCR is like looking for a needle in a haystack, and then making a haystack out of the needle What do you need for PCR? • • • • • What do you need for replication? 1 2 3 4 The PCR Reaction What do you need? •Template (the STR you want to amplify for the study) •Sequence-specific primers flanking the target sequence 5’ 3’ Reverse primer 5’ 3’ 3’ 3’ 5’ Forward primer 5’ Target sequence •Nucleotides (dATP, dCTP, dGTP, dTTP) •Magnesium chloride (enzyme cofactor) •Buffer, containing salt •Taq polymerase: Thermus aquaticus

What is DNA Profiling

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