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.