Chapter 9 – DNA-Based Information Technologies

• Recombinant DNA molecules are constructed with DNA from different sources

• Recombinant DNA molecules are created often in nature

• Used in the lab for many purposes

•One is to clone genes

DNA Cloning – making many copies of a segment of DNA by attaching it to a smaller, replicating piece of DNA

Five basic steps in cloning experiment

1. Preparation of DNA to be cloned (target)

Needs to be cut from source.

2. Selection of vector – self-replicating piece of DNA

3. Joining (Ligation) of target and vector DNA fragments. (Use of enzymes)

Product is recombinant DNA

Five basic steps (cont)

4. Introduction of recombinant DNA into compatible host cells and growth therein. Genetic transformation is the uptake of foreign DNA by a host cell

5. Selecting & Identifying of host cells that contain recombinant DNA of interest. (screening)

Cutting of target and vector DNA uses Nucleases

• Nucleases - hydrolyze phosphodiester bonds RNases (RNA substrates) DNases (DNA substrates)

• May cleave either the 3’- or the 5’- ester bond of a 3’-5’ phosphodiester linkage

• Exonucleases start at the end of a chain

• Endonucleases hydrolyze sites within a chain

• Nuclease cleavage sites

• Cleavage at bond A generates a 5’-phosphate and a 3’ OH terminus

• Cleavage at bond B generates a 3’-phosphate and a 5’-hydroxyl terminus

Restriction Endonucleases

• Enzymes that recognize specific DNA sequences

• Cut both strands of DNA at the binding site, producing fragments that can be degraded by exonucleases

• Host cells protect their own DNA by covalent modification of bases at the restriction site (e.g. methylation)

Restriction endonuclease properties

• Type I - catalyze both the methylation of host DNA and cleavage of unmethylated DNA at a specific recognition sequence

• Type II - cleave double-stranded DNA only, at or near an unmethylated recognition sequence

• More than 200 type I and type II are known

• Most recognize “palindromic sequences” (read the same in either direction)

Nucleases leave either:

sticky ends- staggered cut leaves an overhang of ssDNA on each strand

blunt ends – cut directly across both strands leaving all dsDNA

Cutting with EcoRI

Cloning Vectors

• Cloning vectors can be: plasmids, bacteriophages, viruses, small artificial chromosomes

• Vectors have at least one unique cloning site: a sequence cut by a restriction endonuclease to allow site-specific insertion of foreign DNA

• Restriction enzymes can generate recombinant DNA

Plasmid Vectors

• Plasmids are small, circular DNA molecules used as vectors for DNA fragments to 20kb

• Replicate autonomously within a host cell

• Carry genes conferring antibiotic resistance, used as marker genes for cells carrying vectors

• pBR322 was one of the first plasmid vectors

Plasmid vector pBR322

• pBR322 has 4361 base pairs

• Origin of replication (ori)

• Antibiotic resistance genes amp and tet

• Rop gene regulates replication for ~20 copies of the plasmid per cell

B. Bacteriophage Vectors

• Efficient, commonly used vector for delivering DNA into a bacterial cell

• Advantage over plasmid vectors is that transfection is more efficient than transformation

• Disadvantage: DNA must be packaged into phage particles in vitro

C. Yeast Artificial Chromosomes

as Vectors• Large DNA fragments can be inserted

into artificial chromosomes that are replicated in eukaryotic cells

• Such chromosomes must be linear and contain a eukaryotic replication origin

• Yeast artificial chromosome (YAC)

Yeast artificial chromosome (YAC)

D. Bacterial Artificial Chromosomes (BAC’s)

• Special Plasmids designed for cloning large DNA fragments (100-300 kbp)

Identification of Host Cells Containing Recombinant

DNA• After a cloning vector and insert DNA have been

joined in vitro, recombinant DNA is introduced into a host cell such as E. coli (transformation)

• Only a small percentage of cells take up the DNA

• Selection -cells are grown under conditions in which only transformed cells survive

• Screening - transformed cells are tested for the presence of the recombinant DNA

A. Selection Strategies Use Marker Genes

• Bacterial plasmid vectors can carry a -lactamase marker gene (marker genes allow detection of cells)

• -Lactamase hydrolyzes -lactam antibiotics (e.g. ampicillin)

• Only cells transformed with plasmids expressing the -lactamase gene are ampicillin resistant and can grow in media containing ampicillin (ampR)

Selection or screening by insertional activation

• Insertional inactivation - insertion of a DNA fragment within the coding region of a gene on a vector results in inactivation of that gene

• If the gene product can be detected, this can be used for selection and screening

• bBR322 gene for tetracycline resistance (tetR) can be inactivated by DNA insertion making them tetracycline sensitive (tetS)

Visual Markers: Insertional Inactivation of the -Galactosidase

Gene• The lacZ gene of E. coli encodes -galactosidase

and cleavage of an artificial substrate produces a blue dye (X-gal)

• Vectors without inserts in the lacZ gene give rise to blue colonies in the presence of X-gal

• Vectors with DNA inserted in the lacZ gene do not produce the enzyme and yields colonies which are white

Blue/white screen

• Blue colonies: cells transformed with cloning vectors not containing inserts(-galactosidase is active)

• White colonies: cells transformed with recombinants. -Galactosidase gene disrupted by insert

Genomic Libraries

• A method for isolating large quantities of specific DNA fragments from organisms

• DNA library consists of all the recombinant DNA molecules generated by ligating all the fragments of a particular DNA into vectors

• Recombinant DNA molecules are then introduced into cells for replication

Genomic library properties

• Genomic libraries represent all the DNA from an organism’s genome

• Partial (rather than total) restriction digestion is used to ensure that every gene is represented

• All types of vectors used

• Genomic libraries include both expressed and non-expressed DNA from the organism

cDNA Libraries Are Made from Messenger RNA

• cDNA libraries represent all the mRNAs made in a given cell or tissue

• cDNA (complementary DNA) is double-stranded DNA made with reverse transcriptase

• Purification of mRNA relies on the polyA tails on mature eukaryotic mRNA

• The more abundant rRNA and tRNA lack tails

Preparation of cDNA

Properties of cDNA libraries

• Using a cDNA library from a specific tissue with abundant protein of interest increases the chances of successfully cloning the gene for that protein

• Specialized phage l vectors and plasmids are used in constructing cDNA libraries

• cDNA libraries from mRNA do not include introns or flanking sequences (much less complex than genomic libraries)

Expression of Proteins Using Recombinant DNA

Technology

• Cloned or amplified DNA can be purified and sequenced or used to produce RNA and protein

• Such DNA can also be introduced into organisms to change their phenotype

• Purification of proteins begins with overproduction of the protein in a cell containing the expression vector

A. Prokaryotic Expression Vectors

• Expression vectors - plasmids that have been engineered to contain regulatory sequences for transcription and translation

• Eukaryotic genes can be expressed in prokaryotes

• Expression of a eukaryotic protein in E. coli

B. Expression of Proteins in Eukaryotes

• Prokaryotic cells may be unable to produce functional eukaryotic genes

• Some expression vectors are for eukaryotes

• Recombinant DNA molecules can also be integrated into the genomes of large multicellular organisms

• Creates transgenic organisms

• Technique for creating a transgenic mouse

Effect of an extra growth hormone gene in mice

• Transgenic mouse (left) carries a gene for rat growth hormone

• Normal mouse (right)