Recombinant dna technology

Recombinant DNA Technology

Anas Bahnassi PhD RPh CDE CDM

The Role of Recombinant DNA Technology in Biotechnology

• Recombinant DNA Technology

– Intentional modification of organisms’ genomes for practical purposes

– Three goals

• Eliminate undesirable phenotypic traits

• Combine beneficial traits of two or more organisms

• Create organisms that synthesize products humans need

Overview of

recombinant

DNA

technology

Bacterial cell

Bacterial chromosome

Plasmid

Gene of interest

DNA containing gene of interest

Isolate plasmid.

Enzymatically cleave DNA into fragments.

Isolate fragment with the gene of interest.

Insert gene into plasmid.

Insert plasmid and gene into bacterium.

Culture bacteria.

Harvest copies of

gene to insert into

plants or animals

Harvest proteins coded by gene

Eliminate

undesirable

phenotypic

traits

Produce vaccines,

antibiotics,

hormones, or

enzymes

Create

beneficial

combination

of traits

The Tools of Recombinant DNA Technology

• Mutagens

– Physical and chemical agents that produce mutations

– Scientists utilize mutagens to

• Create changes in microbes’ genomes to change phenotypes

• Select for and culture cells with beneficial characteristics

– Mutated genes alone can be isolated


• The Use of Reverse Transcriptase to Synthesize cDNA

– Isolated from retroviruses

– Uses RNA template to transcribe molecule of cDNA

– Easier to isolate mRNA molecule for desired protein first

– mRNA of eukaryotes has introns removed

• Allows cloning in prokaryotic cells


• Synthetic Nucleic Acids

– Molecules of DNA and RNA produced in cell-free solutions

– Uses of synthetic nucleic acids

• Elucidating the genetic code

• Creating genes for specific proteins

• Synthesizing DNA and RNA probes to locate specific sequences of nucleotides

• Synthesizing antisense nucleic acid molecules


• Restriction Enzymes

– Bacterial enzymes that cut DNA molecules only at restriction sites

– Categorized into two groups based on type of cut

• Cuts with sticky ends

• Cuts with blunt ends

Actions of restriction

enzymes-overview

Origin and function

• Bacterial origin = enzymes that cleave foreign DNA

• Named after the organism from which they were derived

– EcoRI from Escherichia coli

– BamHI from Bacillus amyloliquefaciens

• Protect bacteria from bacteriophage infection

– Restricts viral replication

• Bacterium protects it’s own DNA by methylating those specific sequence motifs

Availability

• Over 200 enzymes identified, many available commercially from biotechnology companies

Classes

• Type I

– Cuts the DNA on both strands but at a non-specific location at varying distances from the particular sequence that is recognized by the restriction enzyme

– Therefore random/imprecise cuts

– Not very useful for rDNA applications

Classes

• Type II – Cuts both strands of DNA within the particular sequence

recognized by the restriction enzyme – Used widely for molecular biology procedures – DNA sequence = symmetrical

Classes

• Reads the same in the 5’ 3’ direction on both strands = Palindromic Sequence

• Some enzymes generate “blunt ends” (cut in middle)

• Others generate “sticky ends” (staggered cuts) – H-bonding possible with complementary tails

– DNA ligase covalently links the two fragments together by forming phosphodiester bonds of the phosphate-sugar backbones


• Vectors

– Nucleic acid molecules that deliver a gene into a cell

– Useful properties

• Small enough to manipulate in a lab

• Survive inside cells

• Contain recognizable genetic marker

• Ensure genetic expression of gene

– Include viral genomes, transposons, and plasmids

Producing a

recombinant

vector

Antibiotic resistance gene

Restriction site

mRNA for human growth hormone (HGH)

Reverse transcription

Plasmid (vector)

cDNA for HGH

Restriction enzyme

Restriction enzyme

Sticky ends

Gene for human growth hormone

Ligase

Recombinant plasmid

Introduce recombinant plasmid into bacteria.

Recombinant plasmid

Bacterial chromosome

Inoculate bacteria on media containing antibiotic.

Bacteria containing the plasmid with HGH gene survive because they also have resistance gene.

Requirements of a vector to serve as a carrier molecule

• The choice of a vector depends on the design of the experimental system and how the cloned gene will be screened or utilized subsequently

• Most vectors contain a prokaryotic origin of replication allowing maintenance in bacterial cells.


• Some vectors contain an additional eukaryotic origin of replication allowing autonomous, episomal replication in eukaryotic cells.

• Multiple unique cloning sites are often included for versatility and easier library construction.


• Antibiotic resistance genes and/or other selectable markers enable identification of cells that have acquired the vector construct.

• Some vectors contain inducible or tissue-specific promoters permitting controlled expression of introduced genes in transfected cells or transgenic animals.


• Modern vectors contain multi-functional elements designed to permit a combination of cloning, DNA sequencing, in vitro mutagenesis and transcription and episomal replication.

Main types of vectors

• Plasmid, bacteriophage, cosmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), yeast 2 micron plasmid, retrovirus, baculovirus vector……

Choice of vector

• Depends on nature of protocol or experiment

• Type of host cell to accommodate rDNA

– Prokaryotic

– Eukaryotic

Plasmid vector

• Covalently closed, circular, double stranded DNA molecules that occur naturally and replicate extrachromosomally in bacteria

• Many confer drug resistance to bacterial strains

• Origin of replication present (ORI)


• Gene Libraries

– A collection of bacterial or phage clones

• Each clone in library often contains one gene of an organism’s genome

– Library may contain all genes of a single chromosome

– Library may contain set of cDNA complementary to mRNA

Production of a

gene library-

overview

Genome

Isolate genome or organism.

Generate fragments using restriction enzymes.

Insert each fragment into a vector.

Introduce vectors into cells.

Culture recombinant cells; descendants are clones.

Definition of recombinant DNA

• Production of a unique DNA molecule by joining together two or more DNA fragments not normally associated with each other

• DNA fragments are usually derived from different biological sources

Steps involved in isolating a particular DNA fragment from a complex mixture of DNA fragments or

molecules

1. DNA molecules are digested with enzymes called restriction endonucleases which reduces the size of the fragments Renders them more manageable for cloning purposes


molecules

2. These products of digestion are inserted into a DNA molecule called a vector Enables desired fragment to be replicated in cell culture to very high levels in a given cell (copy #)


molecules

3. Introduction of recombinant DNA molecule into an appropriate host cell – Transformation or transfection – Each cell receiving rDNA = CLONE – May have thousands of copies of rDNA molecules/cell

after DNA replication – As host cell divides, rDNA partitioned into daughter cells


molecules

4. Population of cells of a given clone is expanded, and therefore so is the rDNA. – Amplification – DNA can be extracted, purified and used for molecular analyses

• Investigate organization of genes • Structure/function • Activation • Processing

– Gene product encoded by that rDNA can be characterized or modified through mutational experiments

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Anas Bahnassi PhD RPh

Pharmaceutical Biotechnology

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Recombinant dna technology

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