Chapter 20
DNA Technology & Genomics
O.J. Simpson capital murder case
• 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. BrownSimpson, 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 U.S.: 2.8 million to 1
• Odds of winning Illinois Big Game 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 having quintuplets: 85 million to 1
• Odds of being struck by a meteorite: 10 trillion to 1
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 reproducible way
• Sticky end:
– short 1-sided 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 or Yeast Artificial Chromsomomes)
– YAC’s are small euk chromosomes that can hold genes & be inserted
into euks and behave normally
Restriction Enzymes
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, electroporation in euks)
• Cloning of cells (and foreign genes)
• Identification of cell clones carrying the gene of interest
Genomic Library
• Stores cloned genes
– In a large plasmid called a Bacteria Artificial
Chromosome (BAC)
– Also can use a cDNA library
• Uses mRNA reverse transcribed into cDNA
• More limited – excludes introns
• cDNA = complementary DNA
DNA Analysis & Genomics
• PCR (polymerase chain reaction)
• Gel electrophoresis
• Restriction fragment analysis
(RFLPs)
• Southern blotting
• DNA sequencing
• Human genome project
Polymerase chain reaction (PCR)
• Amplifies DNA piece w/out
cells (in vitro)
• Materials:
– heat,
– DNA polymerase,
– nucleotides,
– single-stranded DNA
primers
• Applications:
– fossils, forensics,
prenatal diagnosis, etc.
DNA Analysis
• Gel electrophoresis: separates nucleic acids or
proteins on the basis of size or electrical charge
creating DNA bands of the same length
Video: Biotechnology Lab
Restriction fragment analysis
• Restriction fragment length polymorphisms (RFLPs)
• Southern blotting: process that reveals sequences
and the RFLPs in a DNA sequence
– Cut, gel electrophoresis, and marking/analyzing
pieces
• DNA Fingerprinting
• For mRNA similar process called Northern Blot
• For Protein called Western Blot
Gene Expression
• RT-PCR: PCR of cDNA throughout development
– Use fluorescent dyes with probes to light up expressed gene
and location/timing
– Can be in situ-hybridization: in the intact organism
• DNA Microarray Assays
– Tiny amounts of a large number of single-stranded DNA gene
fragments on a glass slide in an array (grid)
– Slide exposed to cDNA molecules made from mRNAs in 2
different cells of interest and labeled with fluorescent dyes
– Expressed genes hybridize and fragment changes color
– Colored lasers “read” expression: red 1, green 2, yellow both
DNA Microarray Assays
Study Expression Effects
• Turn off gene to see what effect is (what gene does)
– in vitro mutagenesis damages a gene in a cell
• Prevents expression due to damage
– RNAi (RNA Interference)
• Uses synthetic RNA to damage specific mRNA
• Newer tech, but has reduced translation successfully
Practical DNA Technology Uses
• Diagnosis of disease
• Human gene therapy
• Pharmaceutical products
(vaccines)
• Forensics
• Animal husbandry
(transgenic organisms)
• Genetic engineering in
plants
• Stem cells
• Ethical concerns?
Chapter 21
Genomes and Their
Evolution
Overview
• Many organisms have had their whole genome
sequenced
• Genomics now can compare genomes of organisms
for info about evolutionary history of genes
• Bioinformatics is a new field using computers to
storage and analyze all this bio data
Bioinformatic Resources
• NCBI database of sequences: Genbank
– Over 100 billion base pairs and growing fast
• NCBI sodtware BLAST: allows visitors to compare a
DNA sequence to every sequence in Genbank
• Other programs allow comparing predicted protein
sequences or searching protein sequences for an
amino acid sequence and makes a 3D model of it
• Stores all known 3D protein structures
Identifying Protein-Coding Genes
• Computer analysis of genome sequences helps
identify sequences likely to encode proteins
– Comparison of sequences of “new” genes with
those of known genes in other species
• Proteomics is the systematic study of all proteins
encoded by a genome
• Knowing the genes can help create accurate assays
• Systems level biologists then analyze differences in
expression using Gene Chips with all human genes
to evaluate disease/cancer causes and effects
Gene Density & Noncoding DNA
• Number of genes is not correlated to genome size
• Vertebrate genomes can produce more than one
polypeptide per gene (alternative splicing of RNA)
• Mammals have the lowest gene density
Fig. 21-7
Exons (regions of genes coding for protein
or giving rise to rRNA or tRNA) (1.5%)
Repetitive
DNA that
includes
transposable
elements
and related
sequences
(44%)
L1
sequences
(17%)
Introns and
regulatory
sequences
(24%)
Unique
noncoding
DNA (15%)
Repetitive
DNA
unrelated to
transposable
elements
(15%)
Alu elements
(10%)
Simple sequence Large-segment
DNA (3%)
duplications (5–6%)
Building a Genome
• First cells likely had few genes
• Genes can be added: polyploidy/duplication
– Copies can then change (i.e. -globin and -globin)
– Can be inversed or otherwise rearranged
– Some “hotspots” are more likely to break/alter
– Differences can help speciation
Transposable Elements
• Allow recombination between different chromosomes
• May block protein production (when inserted in exon)
• May increase or decrease protein production (when
inserted in regulatory sequence)
• May carry a gene or groups of genes to a new location
• May also create new sites for alternative splicing in an
RNA transcript
• Changes are usually detrimental but may on occasion
prove advantageous for evolution
Fig. 21-17
Adult
fruit fly
Fruit fly embryo
(10 hours)
Fly
chromosome
Mouse
chromosomes
Mouse embryo
(12 days)
Adult mouse