General Biology/Genetics/Recombinant DNA Technology

=Recombinant DNA technology =
 * Revolutionized modern biology
 * Ability to manipulate genes in vitro
 * Hybrid genes, including combining genes of different species
 * Detailed study of gene function
 * Determine nucleotide sequences of genes and their regulators (deduce amino acid sequences of proteins)
 * Genome projects: complete nucleotide sequence of >40 genomes, including human
 * Made possible by convergence of:
 * discovery of restriction enzymes
 * genetics of bacteria and their plasmids

Recombinant DNA technology

 * Uses
 * Detailed study of gene function
 * Homeostasis, response to stress
 * Development (birth defects)
 * Evolution of genes informs on evolution of life
 * Human betterment
 * Medicine
 * Identification, treatment of genetic disorders
 * Molecular medicine: from deduced amino acid sequences, design better drugs
 * Foods
 * Improve crop yield, resistance to disease
 * Improve nutritional value
 * Forensics
 * DNA fingerprinting: guilt or innocence

Restriction endonucleases
Originally found in bacteria to prevent invasion of viral DNA, cuts double stranded DNA that is unmethylated, will not cut newly synthesized DNA since hemi-methylated, a product of semi-conservative replication of DNA
 * sever phosphodiester bonds of both polynucleotide strands in order to combine foreign DNA
 * create restriction fragments (restriction digestion)
 * 5’ phosphate and 3’ –OH at ends
 * usually nucleotide specific target sequence
 * 4-6 bp most common, the more bases, then the more specific for recombination
 * cuts in or near sequence
 * ends
 * sticky=overhanging ends, 5’ or 3’
 * blunt ends - straight cut, will anneal with any other blunt end in the presence of high ligase
 * Hundreds of know restriction endonucleases, usually named after the bacteria that it was found in
 * e.g. EcoR1, Alu1, BamHI, HindIII

Restriction endonucleases
Gene cloning
 * Cloning:
 * Restriction digestion of DNA
 * insertion of restriction fragment into cloning vector
 * Bacterial plasmid
 * Bacterial virus
 * Yeast artificial chromosomes
 * Transformation of bacteria with recombinant plasmid, virus
 * Screening for clone of interest by using reporter genes or resistance upon exposure to anti-biotic

Uses of cloned gene

 * Determine nucleotide sequence and deduce amino acid sequence from genetic code
 * Submit to GenBank (available on WWW)
 * Manipulate gene to study function
 * In vitro
 * In vivo
 * Transgenic (recombinant) organisms
 * Knockout organisms
 * Medical and commercial uses

Other molecular procedures

 * Polymerase chain reaction (Mullis)
 * Amplifies target DNA without cloning
 * Target amount can be single molecule
 * Amplified DNA can be sequenced, cloned, etc.
 * Southern blotting
 * Used to identify restriction fragments carrying particular gene
 * Also used for DNA fingerprinting and RFLP analysis
 * cDNA construction
 * Reverse transcription from mRNA template

RFLP(restriction fragment length polymorphism) analysis

 * Basis of DNA fingerprinting using SNP - single nucleotide polymorphisms and repeats of DNA sequence
 * Many uses
 * Criminal cases using multiple probes
 * Parentage
 * Species identification
 * Gene evolution
 * Species evolution

Sanger DNA sequencing

 * Uses dideoxynucleotides (ddNTP), a template strand, DNA polymerase 1 (Also known as Kornberg enzymes) and dNTPs
 * Missing 3’-OH for nulceopjilic attack for elongation
 * DNA synthesis stops after one is incorporated into DNA fragment
 * ratio of ddNTP to dNTP determines likelihood of termination
 * Manual method with 32P-labeled ddATP and 4 test tubes - ddATP, ddCTP, ddGTP, ddTTP
 * Automated method using ddNTPs labeled with fluorescent dyes in capillary tube
 * Often done commercially

Automated sequencing
Typical machine
 * 2 hour sequencing run
 * 600-1000 bases per sample
 * multiple samples
 * Up to 500,000 bases per day (12 hr)
 * Data processed by computer
 * In big labs, sequencing reactions also are automated

Genome projects

 * Determine entire nucleotide sequence of genome
 * >40 genomes sequenced
 * Helicobacter pylori
 * Escherichia coli
 * Saccharomyces cerevisiae
 * Caenorhabditis elegans
 * Drosophila melanogaster
 * Homo sapiens (first rough draft)
 * Computer identifies all genes, based on properties of genes (e.g., start/stop codons, introns, etc.).

Biochips
Microarray chips contain wells of DNA that code for specific genes that uses the concept of hybridization with the gene of interest to see if a gene is expressed or is present.
 * Microarray of DNA fragments, size of postage stamp; can be expensive, but has decreased in cost
 * Designed to detect:
 * mutated genes (SNPs)
 * expressed genes
 * Instant DNA profile (“GATTACA”)

DNA chip controversies

 * Medicine
 * Risks and informed consent for gene replacement therapy
 * Alteration of human gene pool
 * Parental choice
 * Privacy
 * Genetically modified foods
 * Safety
 * Labeling
 * Forensics
 * Mandatory tests
 * Reliability standards

Gene patenting

 * Techniques to study and manipulate genes are patented (e.g., cloning and PCR)
 * Should genes be patented?
 * Are they the intellectual property of the discoverer?
 * Don’t they belong to all of us?
 * Should indigenous peoples be compensated for useful genes extracted from their local plants and fungi?

Stem cells

 * Totipotent cells from early embryo
 * grow into any tissue or cell type
 * Recombinant genes can be introduced
 * Considerable use in analyzing gene expression in mice
 * Possible therapeutic use in humans
 * Very controversial

This text is based on notes very generously donated by Paul Doerder, Ph.D., of the Cleveland State University.