DNA Technology and Genomics

Recombinant DNA n Definition: DNA in which genes from 2 different sources are linked n Genetic engineering: direct manipulation of genes for practical purposes n Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products

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

Bacterial DNA-transfer processes n Transformation –genotype alteration by the uptake of naked, foreign DNA from the environment (Griffith expt.) n 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 n Conjugation –direct transfer of genetic material; cytoplasmic bridges –pili; sexual

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

Recombination of E. coli

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

DNA Cloning n Restriction enzymes (endonucleases) –in nature, these enzymes protect bacteria from intruding DNA –they cut up the DNA (restriction) –very specific n Restriction site –recognition sequence for a particular restriction enzyme n Restriction fragments –segments of DNA cut by restriction enzymes in a reproducable way n Sticky end –short extensions of restriction fragments n DNA ligase –enzyme that can join the sticky ends of DNA fragments n Cloning vector –DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids)

Eukaryotic Gene Cloning n Isolation of cloning vector (bacterial plasmid) & gene-source DNA (gene of interest) n Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind fragmented DNA w/ DNA ligase n Introduction of cloning vector into cells (transformation by bacterial cells) n Cloning of cells (and foreign genes) n Identification of cell clones carrying the gene of interest

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

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

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

Polymerase chain reaction (PCR) n Amplification of any piece of DNA without cells (in vitro) n Materials: heat, DNA polymerase, nucleotides, single-stranded DNA primers n Applications: fossils, forensics, prenatal diagnosis, etc.

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

Southern Blotting

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

Genomics n 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

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

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

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

Transposons n 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”

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

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

Multigene Families n 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 n Pseudogenes- nonfunctional nucleotide sequences (very similar to functional genes)

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