DNA Technology and Genomics

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) • 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