1. DNA Technology and Genomics

2. Recombinant DNA
Definition: 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

3. Bacterial genetics Nucleoid:
region in bacterium densely packed with DNA (no membrane)
Plasmids:
small circles of DNA
Reproduction by binary fission (asexual)

4. Bacterial DNA-transfer processes
Transformation
genotype alteration by the uptake of naked, foreign DNA from the environment (Griffith expt.)
Transduction
phages that carry bacterial genes from 1 host cell to another
generalized~ random transfer of host cell chromosome
specialized~ incorporation of prophage DNA into host chromosome
Conjugation
direct transfer of genetic material; cytoplasmic bridges
pili; sexual

5. Bacterial Plasmids
Small, circular, self-replicating DNA separate from the bacterial chromosome
F (fertility) Plasmid: codes for the production of sex pili (F+ or F-)
R (resistance) Plasmid: codes for antibiotic drug resistance

6. Recombination of E. coli

7. Bacterial plasmids in gene cloning
Clone genes for insertion into organisms
Clone proteins for medical/ pharmaceutical purposes

8. 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 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)

9. 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 fragmented DNA w/ 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

10. Genomic Libraries
Cloned genes from a genome are stored in a “genomic library”
Recombinant fragments in bacteria or phages
Complimentary DNA (cDNA) Library
mRNA extracted
Reverse transcriptase makes a complimentary strand of gene

11. DNA Analysis PCR (polymerase chain reaction) Gel electrophoresis
Restriction fragment analysis (RFLPs)
Southern blotting
DNA sequencing

12. Practical DNA Technology Uses
Diagnosis of disease
Human gene therapy
Pharmaceutical products
Vaccines
Hormones
Forensics
Crime scene analysis of DNA
Animal husbandry (transgenic organisms)
“Pharm” animals
Genetic engineering in plants
Disease/ pest resistance

13. 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.

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

15. Restriction fragment analysis
Restriction fragment length polymorphisms (RFLPs)
Differences in restriction fragment patterns on homologous chromosomes
Occur in noncoding DNA sequences
Serve as inheritable genetic markers
Southern blotting: process that reveals sequences and the RFLPs in a DNA sequence
DNA Fingerprinting

16. Southern Blotting

17. DNA Sequencing Determination of nucleotide sequences
Dideoxy Chain-Termination Method (Sanger Method)
Whole-genome approach (Venter and Celera Genomics)
Genomics: the study of genomes based on DNA sequences
Human Genome Project
Begun in 1990; largely completed by 2003

18. Genomics The National Center for Biotechnology Information (NCBI)
Created a database of gene sequences created by the Human Genome Project and other sequencing endeavors
Genbank
BLAST software allows for comparison of sequences

19. Analyzing Gene Expression
Northern Blotting
Gel electrophoresis done with labeling probes to determine function
RT-PCR
Uses reverse transcriptase and PCR
Compares gene expression between different samples

20. Studying Gene Interaction
DNA Microassay
Many DNA fragments on a glass slide or chip
Can be tested for interaction with other genes marked with fluorescent markers

21. Determining Gene Function
In vitro mugagenasis
Disable certain genes and observe consequences
Mutations “knock out” certain genes
RNA interference (RNAi)
RNA used to block translation of certain genes

22. Transposons
transposable genetic element; piece of DNA that can move from location to another in a cell’s genome
chromosome to plasmid, plasmid to plasmid, etc.)
“jumping genes”

23. Eukaryotic Genes
98.5% of all DNA does not code for proteins, rRNA, or tRNA
Most is repetitive DNA
44% is made of transposable elements

24. Transposable Elements
Transposons
Move w/in a genome by DNA intermediate
Barbara McClintock (1940’s and 50’s)
“Jumping genes”
Researched the location of colored kernels in maize
Retrotransposons
Move by means of a RNA intermediate

25. Multigene Families
In the human genome, ½ of coding DNA is in multigene families
Collections of identical or very similar genes
Identical- ribosomal RNA molecules
Similar- α-globin and β-globin
Pseudogenes- nonfunctional nucleotide sequences (very similar to functional genes)

26. Genome Evolution Duplications of chromosomes
Unequal crossing over
Duplication and divergence of DNA segments
Ancestral globin gene  present day α-globin and β-globin genes
Rearranging genes
Exon duplication/ exon shuffling
Transposable elements