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Recombinant Chapter DNA Technology
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Some Terminology
genetic engineering
recombinant DNA technology
deliberate modification of organism's genetic information by directly changing the sequence of nucleic acids in its genome procedures used to carry out genetic engineering
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More Terminology
cloning
biotechnology
generation of a large number of genetically identical DNA molecules
use of organisms to form useful products industrial microbiology use of microbes to manufacture important compounds or the use of microbes as products in their own right
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Key Developments in Recombinant DNA Technology
Restrictions enzymes Genetic cloning and cDNA synthesis Southern blotting
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Figure 15.1
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Restriction Enzymes
recognize and bind specific sequences in DNA called recognition sites cleave DNA at this site or a defined distance from it may produce sticky ends or blunt ends in the DNA target hundreds are commercially available
Figure 15.2
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Figure 15.3
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Table 15.2
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Genetic Cloning and cDNA Synthesis
Jackson, Symons, and Berg (1972)
generated first recombinant DNA molecules foreign DNA target can be recombined into plasmid vector plasmid vectors replicate and maintain foreign fragment
vectors carriers of foreign DNA
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cDNA Synthesis and Reverse Transcriptase
synthesizes double stranded DNA from RNA template (mRNA)
used to construct complementary DNA (cDNA) independently discovered by Temin and Baltimore in 1970
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Figure 15.4
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Figure 15.5
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Southern Blotting Technique
Edwin M. Southern (1975) developed for use in detecting specific DNA fragments 3 step process separate DNA molecules transfer separated DNA molecules hybridize to radioactive specific DNA probe autoradiography method for detecting radioactively labeled molecules
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Figure 15.6
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First Recombinant Molecule
1982 genetically cDNA fragment coding
for human insulin recombined into E. coli vector produced new source of human insulin first commercial use for recombinant molecules
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The Polymerase Chain Reaction (PCR)
enables gene amplification
the rapid synthesis of many copies of a specific DNA fragment from a complex mixture of DNA and other cellular components oligonucleotides (DNA or RNA)
chain length can vary from short (230 nucleotides) to long (50100 nucleotides) serve as DNA primers
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PCR Reaction Mix
reaction mix contains
thermocycler is the instrument used
primers target DNA thermostable DNA polymerase such as Taq polymerase each of the four deoxyribonucleotide triphosphates
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More about PCR...
in the reaction
DNA is denatured primers anneal to target DNA copies of the target DNA are synthesized, i.e., amplified
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Figure 15.7
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Uses of PCR
simplifies gene cloning generates DNA fragments for nucleotide sequencing may amplify environmental genes without culturing the microbes diagnostic purposes AIDS, Lyme disease, chlamydia, tuberculosis, hepatitis, human papilloma virus
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Figure 15.8
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Gel Electrophoresis
used to separate molecules based on their charge and molecular weight agarose or acrylamide gels can be used to separate DNA fragments DNA acidic; is it migrates from the negative to the positive end of the gel
each fragment's migration rate is inversely proportional to the log of its molecular weight
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Figure 15.9
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Cloning Vectors and Creating Recombinant DNA
there are four types of cloning vectors
plasmids (most commonly used) phages and viruses cosmids artificial chromosomes
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Table 15.3
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Plasmids
replicate autonomously and easy to purify requirements for vectors
an origin of replication a selectable marker a multicloning site or polylinker
site that allows gene to be cloned to placed into the plasmid vector
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Figure 15.10
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Figure 15.11
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Phage Vectors
are engineered phage genomes previously genetically modified to include restriction sites after insertion of foreign DNA the recombinant phage genome is packaged into the capsid and used to infect host cells
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Cosmids
do not exist in nature these vectors have been constructed to contain features from both phages and plasmids
they have a selectable marker, multiple cloning sites from plasmids, and a cos site from phage phage then introduces recombinant DNA into E. coli
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Artificial Chromosomes
used when large fragments of DNA must be cloned bacterial artificial chromosomes (BACs)
yeast artificial chromosomes (YACs)
played important role in the human genome project may be unstable, for very large DNA fragments
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Figure 15.12
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Construction of Genomic Libraries
used when gene of interest is on a chromosome that has not been sequenced the library is constructed by cleaving the genome and then cloning the fragments into vectors the libraries are screened for the genes of interest in a variety of ways cDNA library may also be constructed for DNA fragments without intron sequences
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Figure 15.13
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Inserting Recombinant DNA into Host Cells
most common hosts
hosts are engineered to lack
E. coli bacteria S.cerevisiae eukaryotic host
restriction enzymes and recA
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Inserting Recombinant DNA into Eukaryotic Host Cells
DNA introduction into microbes
transformation electroporation microinjection gene gun Ti plasmid of Agrobacterium tumefaciens (used to introduce foreign DNA into plant genomes)
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Expressing Foreign Genes in Host Cells
cloned genes, in the new host cell, are called heterologous genes and may not be expressed unless they are modified recombinant gene must have a promoter that host RNA polymerase recognizes differences exist in eukaryotic and bacterial hosts mRNA requires a leader sequence and mRNA modifications, intron removal
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More Foreign Gene Expression...
expression vectors are used to overcome problems with expression of recombinant genes in host cells contain inducible promoters that result in high level transcription
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Purification and Study of Recombinant Proteins
protein purification
fluorescent labeling
isolation of gene product polyhistidine tagging (Histagging) isolation of protein by metal ion resin in vivo expression and regulation gene is coupled to green fluorescent and other new fluorescent peptides that undergo selfcatalyzed light production
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Figure 15.14
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Figure 15.15
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