DNA-Based Information Technologies

• DNA Cloning: The Basics• From Genes to Genomes• From Genomes to Proteomes• Genome Applications and New Products of

Biotechnology

Paul BergStanley N. Cohen & Herbert Boyer, 1970s (p.1119)

DNA Cloning: (recombinant DNA technology or genetic engineering)

1. Cutting DNA at precise location by restriction endonucleases.2. Joining two DNA fragments by DNA ligase.3. Selecting a small molecule of DNA capable of self-replication. DNA segment can be joined to cloning vectors (plasmids or viral DNA) to form recombinant DNA.4. Moving recombinant DNA from the test tube to a host cell for replication.5. Selecting or identifying host cells that contain recombinant DNA.

Restriction Endonucleases and DNA Ligase Yield Recombinant DNA.

Restriction endonucleases:• type II (cut at recognition sites) • type I (cut at >1000 bp away) & type III (cut at 25 bp away) also contain methylase activities

sticky end

Mg2+

Dimeric EcoRV???

Not by EcoRV!!!

WRONG!

EcoR1 cut sticky ends

PvuII cut blunt ends

Restriction digestion:

New DNA sequences can be created by inserting synthetic DNA fragment (linkers) between the ends that are being ligated. An insert with multiple restriction sites is called a polylinker.

Terminal transferase can be used to generate sticky endsfor joining two DNA fragments.

Cloning Vectors Allow Amplification of Inserted DNA Segments

pBR322

Cloning foreign DNA in E. coliwith pBR322 (plasmid): small fragment

Transformationby CaCl2, 0oC > 42oCor by electroporation

Positive clones:ampR gene disrupted by insert

Cloning foreign DNA in E. coliwith bacteriophage ~40 kbp fragment

Cloning foreign DNA with bacterialartificial chromosomes (BACs):large fragment

Positive clones:lacZ gene disrupted by insert

Recombinant DNA Technology

• DNA Cloning: The Basics• From Genes to Genomes• From Genomes to Proteomes• Genome Applications and New Products of

Biotechnology

DNA libraries provide specialized catalogs of genetic information

• Genomic library• cDNA library

Contig: ordering of the clones in a DNA library

Sequence-tagged site (STS) can provide landmarks for genomic sequencing projects.

Isolating a Gene from a Cellular Chromosome

Cloning a gene often requires a DNA library

constructing a cDNA library:(complementary DNA)

Expressed sequence tag (EST):Partial sequences of cDNA libraryat random useful in the mappingof large genomes.

Specialized cDNA library: fusing cDNAs to a marker or reporter gene

Example 1: green fluorescence protein (GFP)

Example 2: epitope tag

Specific DNA sequence can be amplifiede.g., by PCR (polymerase chain reaction):

                                             

Kary Mullis

DNA amplified by PCR can be cloned

Hybridization allows the detection of specific sequences

probe (i.e., labeled DNA or RNA) iscomplementary to the DNA being sought

The Southern blot procedure, as applied toDNA fingerprinting.

Designing a probe to detect the gene for a protein ofknown amino acid sequence.

All 8 will match at least 17 of 20 positions.

Genome sequences provide the ultimate genetic libraries

Human Genome Project

Strategy:

James D. Watson

Human Genome Project, started at late 1980 by 20 centers of six nations (coordinated by NIH/USA), led first by Watson and after 1992 by Collins.The completed sequence of the human genome (3x109 bp)was published in April 2003 (efforts spanning 14 yrs).Joining by Celera Co. (funded in 1997 by Venter) accelerated the process (two years ahead of schedule).

Genomic sequencing timeline

Only <1.4% of our DNA acturally encodes proteins

Recombinant DNA Technology

• DNA Cloning: The Basics• From Genes to Genomes• From Genomes to Proteomes• Genome Applications and New Products of

Biotechnology

Proteome: the complement of proteins expressed by a genome.

Proteomics: a field of investigation evolved from the concept of proteome.

Protein functions:

Phenotypic function: the effect of a protein on the organism.

Cellular function: the network of interactions engaged in by aprotein at the cellular level.

Molecular function: the precise biochemical activity of a protein.

Comparative genomics:Sequence or structural relationships provide information on protein function

Conserved gene order (synteny)in the mouse and human genomes

Cellular expression patterns can reveal the cellular function of a gene.

Methods to detect cellular expression patterns:

• Two-dimensional gel electrophoresis• DNA microarrays (DNA chips)• Protein chips

Ways to make a DNA chip:

• Spot synthesized DNA fragment (nanoliter), by robotic devices, onto a solid surface of the chip.

• Direct synthesize DNA fragment by programmed computer, joining one nucleotide to the next in a photoreaction (photolithography), on the solid surface of the chip. >>

Photolithography

DNA microarrays provide compact libraries for studying genes and their expression.

Enlarged image of a DNA chip: 6200 genes of the yeast genome

Detection of protein-protein interactions helps to define cellular and molecular function

• Comparisons of genome composition (Fig.9.24)

• Purification of protein complexes (IP by Ab x tag)

• Yeast two-hybrid analysis (Fig.9.25)

Use of comparative genomics to identify functional genes:(Proteins P3 and P6 may be functionally related)

The yeast two-hybrid system

Recombinant DNA Technology

• DNA Cloning: The Basics• From Genes to Genomes• From Genomes to Proteomes• Genome Applications and New Products of

Biotechnology

Application of Recombinant DNA Technology

Cloned genes can be expressed using expression vector.

Cloned genes can be altered:e.g., by site-directed mutagenesis

Michael Smith

Yeast is an important eukaryotic host for recombinant DNA:Very large DNA segments can be cloned in yeast artificial chromosomes (YACs).

up to 2 x 106 bp genomic fragmentsisolated by pulse field electrophoresis

Cloning DNA in plant system:

aided by bacterial plant parasites

Cloning in Plants Is Aided by a Bacterial Plant ParasiteAgrobacterium tumefacienswhich contains the large (~200 kbp) Ti plasmid.

Transfer of plasmid to host chromosome relies on the 25 bp repeats and the vir gene products (of Ti plasmid). The vir gene is inducible by thephenolic compound acetosyringone (released by wounded plant cell).

Metabolites produced in Agrobacterium-infected plant cells

by T DNA encoded enzymes, that benefit the bacterium and form a plant tumor.

unusual a.a.growth hormones

A two-plasmid strategy to create a recombinant plant.

A tobacco plant in which the gene for firefly luciferase is expressed

Tomato plants engineered to be resistant to some insect larvae (right)that express protein (by bacterium Bacillus thuringiensis) toxic to moth larvae.

Engineered soybeans resistant to herbicide glyphosate (b)

Cloning in Animal CellsPoints the Way to Gene Therapy

Transfer DNA into animal cells,e.g., liposomes, viral vectors.

Engineered mice (right)expressing human growth hormone

Recombinant DNA Technology Yields New Products and Choice !

基因工程的衝擊 ( 科學月刊十三卷十二期 ),1999

Chao & Cohen

基因工程科技帶給人類社會極大的衝擊 ,堪稱是本世紀生命科學最偉大的革命這個科技不僅提供了深入鑽研生命科學的利器 ,也積極有效地應用在醫藥和農業生產 ,為生物科技的實際應用開始了光明前景 .