Chapter 20: Biotechnology

O.J. Simpson capital murder case,1/95-9/95 Odds of blood in Ford Bronco not being R. Goldman’s: 6.5 billion to 1 Odds of blood on socks in bedroom not being N. Brown-Simpson’s: 8.5 billion to 1 Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J. Simpson: 21.5 billion to 1 Number of people on planet earth: 6.1 billion Odds of being struck by lightning in the U.S.: 2.8 million to 1 Odds of winning the Powerball lottery: 76 million to 1 Odds of getting killed driving to the gas station to buy a lottery ticket 4.5 million to 1 Odds of seeing 3 albino deer at the same time: 85 million to 1 Odds of having quintuplets: 85 million to 1 Odds of being struck by a meteorite: 10 trillion to 1

How do crime scene investigators create a DNA profile? 1. Evidence is collected at the crime scene: Blood Tissue Semen Urine Hair Teeth Saliva Bone

2. DNA is extracted from sources at the crime scene and from victim and suspects How do crime scene investigators create a DNA profile?

Since humans are 99.9% identical where do crime scene investigators look for differences in DNA profiles? 3. Crime Scene Investigators search in areas of the genome that are unique from individual to individual and are “anonymous” (control no known trait or function) The areas examined are Short Tandem Repeats or STR’s STR region

Example of an STR: TH01 The TH01 locus contains 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.

Determining genotypes for individuals using STRs 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

To determine the genotype (DNA profile) Crime Scene Investigators make billions of copies of the target sequence using PCR Starting DNA Template 5’ 3’ Target DNA

What’s the point of PCR? PCR, or the polymerase chain reaction, makes copies of a specific piece of DNA PCR allows you to look at one specific piece of DNA by making copies of *only* that piece of DNA PCR is like looking for a needle in a haystack, and then making a haystack out of the needle

Statistics of Chance: M&M Locus 6 Possible Alleles: Green Red Yellow Blue Brown Orange

Probabilities One allele from each parent means 2 copies of gene/locus X= 1 36 Frequency of any M&M genotype

Real-World Probabilities Forensics labs use 13 different loci with multiple alleles Allele frequencies DO NOT follow mathematical principles - allele frequencies vary by population. These 13 loci allow for discrimination of any two people in the world (with the exception of identical twins), living or dead. Probability of a random match when all 13 loci typed: ~1 in 3 trillion.

Butler et al 2003 J Forensic Sci. Allele Caucasians n=302 African American n=258 Latinos n= TH01 Published Allele Frequencies by Population

DNA Analysis Gel electrophoresis: separates nucleic acids or proteins on the basis of size or electrical charge creating DNA bands of the same length

Restriction fragment analysis Restriction fragment length polymorphisms (RFLPs) Southern blotting: process that reveals sequences and the RFLPs in a DNA sequence DNA Fingerprinting

Recombinant DNA Def: DNA in which genes from 2 different sources are linked Genetic engineering: direct manipulation of genes for practical purposes Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products

Bacterial plasmids in gene cloning

DNA Cloning Restriction enzymes (endonucleases):in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific Restriction site: recognition sequence for a particular restriction enzyme Restriction fragments: segments of DNA cut by restriction enzymes in a reproducable way Sticky end: short extensions of restriction fragments DNA ligase: enzyme that can join the sticky ends of DNA fragments Cloning vector: DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids)

Steps for eukaryotic gene cloning Isolation of cloning vector (bacterial plasmid) & gene- source DNA (gene of interest) Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase Introduction of cloning vector into cells (transformation by bacterial cells) Cloning of cells (and foreign genes) Identification of cell clones carrying the gene of interest

DNA Analysis & Genomics PCR (polymerase chain reaction) Gel electrophoresis Restriction fragment analysis (RFLPs) Southern blotting DNA sequencing Human genome project

Polymerase chain reaction (PCR) Amplification of any piece of DNA without cells (in vitro) Materials: heat, DNA polymerase, nucleotides, single-stranded DNA primers Applications: fossils, forensics, prenatal diagnosis, etc.

DNA Sequencing Determination of nucleotide sequences (Sanger method, sequencing machine) Genomics: the study of genomes based onDNA sequences Human Genome Project

Practical DNA Technology Uses Diagnosis of disease Human gene therapy Pharmaceutical products (vaccines) Forensics Animal husbandry (transgenic organisms) Genetic engineering in plants Ethical concerns?