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
1. Evidence is collected at the crime scene:
How do crime
scene investigators
create a DNA
profile?
Blood
Teeth
Tissue
Semen
Hair
Saliva
Urine
Bone
How do crime
scene
investigators
create a DNA
profile?
2. DNA is extracted from sources at
the crime scene and from victim and
suspects
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
Target DNA
To determine the
genotype (DNA
profile) Crime
Scene Investigators
make billions of
copies of the
target sequence
using PCR
5’
3’
3’
5’
Starting DNA
Template
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
1
6
X
1
6
=
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.
TH01 Published Allele Frequencies by Population
Allele
Caucasians
5
n=302
.002
6
.232
.124
.214
7
.190
.421
.279
8
.084
.194
.096
9
.114
.151
.150
10
.008
.002
.014
11
.002
Butler et al 2003 J Forensic Sci.
African
American
n=258
Latinos
n=140
.004
www.cstl.nist.gov/biotech/strbase/pub_pres/Butler2003a.pdf
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) & genesource 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
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 on DNA
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?