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Recombinant DNA Technology Recombinant DNA Technology

Key Methods Key Concepts

1. Cutting DNA Two key properties of nucleic acids


2. Pasting DNA
3. Engineering Recombinant DNA ACGT
Complementary
4. Making DNA from mRNA TGCA
5. Copying DNA
5’ 3’
6. Determining nucleic acid length
7. Sequencing DNA ACGT Antiparallel
8. Probing to identify a gene of interest TGCA
3’ 5’

Recombinant DNA Technology Recombinant DNA Technology


Key Concepts Cutting DNA
How to cut DNA
Property of Protein:nucleic acid interactions • Physical shearing
Proteins
– Random sites

• Enzymatic digesting
– Endonuclease
digestion: site specific
– Restriction
Sequence specific binding Endonucleases

Proteins bind to specific DNA sequences

Restriction Endonuclease Engineering Recombinant DNA


Digestion
EcoRI

• Palindrome (Rotational symmetry)


• Cuts
– Blunt/flush -Double stranded Blunt ends
Cutting DNA
– Staggered -Single stranded “sticky ends”
• 3’ overhang
• Sequence specific
• 5’ overhang
• enzymatic

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Restriction Endonuclease Recombinant DNA Technology
Digestion Cutting and Pasting DNA
Enzymology

Cutting

Restriction Pasting
Endonuclease DNA Ligase
Digests DNA ligates DNA
Proteins (Enzymes) can cut and paste DNA

Engineering Recombinant DNA Engineering Recombinant DNA


Carrier DNA Source DNA

Three Steps
Restriction • 1. Cut source and vector DNA
Endonuclease
cuts DNA – Restriction Endonuclease Digestion

• 2. Insert source fragment into vector


– Hybridization/Ligation
Fragments
Joined:
Hybridization • 3. Put recombinant vector into host
Followed by Ligation
– Transformation

Engineering Recombinant DNA Insert source fragment


into vector

• Hybridization (nonenzymatic)
Vector DNA – Sticky ends (complementary and antiparallel)
– Origin of replication – Salt and temperature
– Capable of independent replication in a
host • Ligation (enzymatic)
– Capable of incorporating DNA – DNA ligase – create phosphodiester bonds complete
– DNA sequence contains unique restriction covalently joined sugar-phosphate backbones
site

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Engineering Recombinant DNA Recombinant DNA Technology
Copying DNA
Vector DNA
Source DNA

Properties of DNA replication:


polymerization

Cloning PCR Amplification


in a host organism in a test tube
Recombinant DNA
Proteins (Enzymes called polymerases)
can make copies of DNA

Recombinant Molecules: Recombinant


Molecules:
Cloning Cloning

• In a host cell (Bacterial cells)


• Insert: range of sizes up to a few kb
• Cloning Vector: accessory chromosome
• Choice of vectors
• Choice of entry method into cell
• Replication in cell
• Recovery from cell

Recombinant Molecules: Recombinant Molecules:


Cloning Vectors Cloning Vectors

Plasmids
• Small circular
• Many copies per cell
• Replicate independently • Means of identifying the recombinant vector
• Convenient restriction sites • Means of recovery of recombinant vector
• Unique (single cut) restriction sites • Choice depends on size of insert

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Vectors
Bacteriophage vectors
• Single stranded
• Double stranded
• Size of insert limited
• Dispensible sequence can
be replaced with insert
sequence
• Headful packaging limits
insert size