1. DNA Technology & Genomics
Chapter 20
DNA Technology & Genomics

2. O.J. Simpson capital murder case
Odds of blood in Ford Bronco not being R. Goldman’s: billion to 1
Odds of blood on socks in bedroom not being N. Brown-Simpson’s: billion to 1
Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J. Simpson: 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

3. 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

4. Bacterial plasmids in gene cloning

5. DNA Cloning Restriction enzymes (endonucleases): Restriction site:
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

6. Restriction Enzymes

7. 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, electroporation in euks)
Cloning of cells (and foreign genes)
Identification of cell clones carrying the gene of interest

9. 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

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

11. 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.

12. Video: Biotechnology Lab
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

13. 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

14. 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

15. DNA Microarray Assays

16. 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

17. 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?

18. Genomes and Their Evolution
Chapter 21
Genomes and Their Evolution

19. 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

20. 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

21. 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

22. 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

23. Repetitive DNA that includes transposable elements and related
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%)
Introns and
regulatory
sequences
(24%)
Unique
noncoding
DNA (15%) L1
sequences
(17%)
Figure 21.7 Types of DNA sequences in the human genome
Repetitive
DNA
unrelated to
transposable
elements
(15%)
Alu elements
(10%)
Simple sequence
DNA (3%)
Large-segment
duplications (5–6%)

24. 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

25. 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

26. Adult fruit fly Fruit fly embryo (10 hours) Fly chromosome Mouse
Fig
Adult
fruit fly
Fruit fly embryo
(10 hours)
Fly
chromosome
Mouse
chromosomes
Figure Conservation of homeotic genes in a fruit fly and a mouse
Mouse embryo
(12 days)
Adult mouse