Biological engineering The recombinant DNA technique Recombinant DNA Any DNA molecule formed by joining DNA fragments from different sources. Commonly produced by cutting DNA molecules with restriction enzymes and then joining the resulting fragments from different sources with DNA ligase. Recombination Any process in which chromosomes or DNA molecules are cleaved and the fragments are rejoined to give new combinations. Occurs naturally in cells as the result of the exchange (crossing over) of DNA sequences on maternal and paternal chromatids during meiosis; also is carried out in vitro with purified DNA and enzymes.
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Biological engineeringThe recombinant DNA
technique
Recombinant DNA Any DNA molecule formed by joining DNA fragments from different sources. Commonly produced by cutting DNA molecules with restriction enzymes and then joining the resulting fragments from different sources with DNA ligase.
Recombination Any process in which chromosomes or DNA molecules are cleaved and the fragments are rejoined to give new combinations. Occurs naturally in cells as the result of the exchange (crossing over) of DNA sequences on maternal and paternal chromatids during meiosis; also is carried out in vitro with purified DNA and enzymes.
Restriction enzyme (endonuclease) Any enzyme that recognizes and cleaves a specific short sequence, the restriction site, in double-stranded DNA molecules. These enzymes are widespread in bacteria and are used extensively in recombinant DNA technology.
Restriction endonucleases
Source Microorganism Enzyme* Recognition Site (↓)† Ends Produced
Arthrobacter luteus AluI AG↓CT Blunt
Bacillus amyloliquefaciens H BamHI G↓GATCC Sticky
Escherichia coli EcoRI G↓AATTC Sticky
Haemophilus gallinarum HgaI GACGC+5↓ ‡
Haemophilus influenzae HindIII A↓AGCTT Sticky
Haemophilus parahaemolyticus
HphI GGTGA+8↓ ‡
Nocardia otitiscaviaruns NotI GC↓GGCCGC Sticky
Staphylococcus aureus 3A Sau3AI ↓GATC Sticky
Serratia marcesens SmaI CCC↓GGG Blunt
Thermus aquaticus TaqI T↓CGA Sticky
Vectors
Cloning vector: An autonomously replicating genetic element used to carry a cDNA or fragment of genomic DNA into a host cell for the purpose of gene cloning.
Commonly used vectors are bacterial plasmids and modified bacteriophage genomes.
The life cycle of bacteriophage
Shuttle vectors allow DNA to be transferred between two different species. The shuttle vector has two origins of replication, allowing replication to occur in either system/host; it "shuttles" between two different species. Typically, one host is bacterial (e.g. E. Coli) and the other host is a eukaryotic organism (e.g. human). The bacterial host is used for all of the cloning steps and the eukaryotic host can be used to study the expression from that cloned gene or can be used to synthesize a product from the gene.
Cloning into a Plasmid
PCR – In vitro amplification of DNA
PCR (polymerase chain reaction) Technique for amplifying a specific DNA segment in a complex mixture by multiple cycles of DNA synthesis from short oligonucleotide primers followed by brief heat treatment to separate the complementary strands
