Chapter 4: recombinant DNA Restriction enzyme analysis Cloning in E. coli plasmids Transformation Biomedical application

Restriction enzymes Restriction enzymes cut double-strand DNA at specific recognition sequences which are often 4-6 base pair palindromes = 5’-3’ sequence is identical on both DNA strands Many restriction enzymes cut the two DNA strands at different points which generates complementary single-strand ends = sticky ends (others = blunt ends)

Restriction enzymes BamHI (from B. amyloliquefaciens ) recognizes GGATCC and cuts between the G’s on both strands

Restriction enzymes

Restriction enzymes Restriction enzymes cut DNA into defined pieces, named restriction fragments

Restriction enzymes DNA fragments of different size (e.g. restriction fragments) can be separated according to their size by gel electrophoresis: agarose gel electrophoresis (300 bp - 15 kb) polyacrylamide gel electrophoresis (1-500 b) =PAGE

Gel electrophoresis Molecular weigth marker (band sizes known) to compare sample band sizes

Recombinant DNA Sticky ends formed by restriction enzymes permit circularization or combinations of DNA restriction fragment(s) by complementary base pairing

Recombinant DNA A new combination of DNA can be made by combining restriction fragments Complementary sticky ends can be covalently linked with DNA ligase to form recombinant DNA Blunt end DNA fragments (for example generated by PCR) can also be ligated (but less efficiently)

Recombinant DNA Ligation of vector and insert DNA ligase

Recombinant DNA A vector is a replicating unit that can be opened to insert another DNA fragment Often plasmids are used as vector in bacteria A plasmid is a small self-replicating circular DNA molecule found in bacteria

Recombinant DNA Plasmid vectors have an origin of replication a selectable marker gene (often an antibiotic resistance) a cloning site or multicloning site (MCS)

Transformation by heat shock or electroshock Recombinant DNA Transformation by heat shock or electroshock bacterium transformation Plasmid replication Replicating bacteria form colony

Recombinant DNA R Selection Medium containing antibiotic Plate bacteria on selective medium R Medium containing antibiotic Select for presence of marker

Recombinant DNA molecules DNA fragments + + = = Vector DNA 1 DNA 2 DNA 3 In reality only one or up to millions of fragments Recombinant DNA molecules

Cloning = purification Recombinant DNA Cloning = purification Transform plasmids into bacteria: a cell will replicate only one plasmid type Plate bacteria to form colonies

Genetic engineering Methods of genetic manipulation are named: Recombinant DNA technology Genetic engineering Gene cloning or gene technology Applications include: Isolation of specific genes Production of specific proteins

Genetic engineering GMO = genetically modified organism, GMM = genetically modified micro-organisme Genetic modification = targeted modification of a genetic characteristic of an organism  transgenic organisme

Biomedical applications Recombinant DNA technology is used to produce large amounts of medically important proteins such as blood clotting factors, insulin,…. In either bacteria, fungi, animal cells, whole animals or plants DNA probes detect mutant genes in hereditary diseases (DNA diagnostics)

Genetic engineering A chimeric gene is constructed of parts of different genes An eukaryotic gene can only be expressed in bacteria when provided with the correct expression signals (and vice versa) Example: human insulin production in bacteria Bacterial promoter Coding region human insulin gene Bacterial terminator

Biomedical applications 1982 Diabetics lack the hormone insulin Initially, insulin was extracted from the pancreas of cows or pigs (different protein) Biotech insulin: safe and easy