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

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Chapter 4: recombinant DNA

Restriction enzyme analysis

Cloning in E. coli plasmids

Transformation

Biomedical application

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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)

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

Restriction enzymes

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Restriction enzymes

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Restriction

enzymes

cut DNA

into defined

pieces,

named

restriction

fragments

Restriction enzymes

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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

Restriction enzymes

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Gel electrophoresis

Molecular weigth marker (band sizes

known) to compare sample band sizes

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Recombinant DNA

• Sticky ends formed

by restriction enzymes

permit circularization or

combinations of DNA

restriction fragment(s)

by complementary

base pairing

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• 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

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Ligation of

vector and insert

DNA ligase

Recombinant DNA

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• 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

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Plasmid vectors have

• an origin of replication

• a selectable marker gene (often an antibiotic resistance)

• a cloning site or multicloning site (MCS)

Recombinant DNA

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Transformation by heat shock or electroshock

bacteriumtransformation

Plasmid replication

Replicating bacteria form colony

Recombinant DNA

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Selection

Plate bacteria on selective medium

Select for presence of marker

Medium containing antibiotic

R

Recombinant DNA

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+

=Vector

DNA fragments

Recombinant DNA molecules

+=

Recombinant DNA

DNA 1DNA 2DNA 3In reality only one or up to millions of fragments

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Cloning = purification

Transform plasmids into bacteria: a cell will replicate only one plasmid type

Plate bacteria to form colonies

Recombinant DNA

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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

Genetic engineering

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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)

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• 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 promoterCoding region human insulin gene

Bacterial terminator

Genetic engineering

• Diabetics lack the hormone insulin

• Initially, insulin was extracted from the pancreas of cows or pigs (different protein)

• Biotech insulin: safe and easy

1982

Biomedical applications