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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 of DNA fragments, size of postage stamp; can be expensive, but has decreased in cost

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.

• 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.