Lecture on Molecular Cloning

Introduction

• Molecular cloning is a key technique in molecular biology and biochemistry.
• Focuses on inserting genetic information (e.g., gene for making a protein) into a plasmid vector and using bacteria to make multiple copies.
• Not about cloning animals but about manipulating genetic info for research.

Key Concepts

Genetic Information and Plasmids

• Genetic sequence: Instructions for making proteins, initially coded in DNA.
• Messenger RNA (mRNA): Copy of the gene, edited to mature mRNA by splicing out introns.
• Complementary DNA (cDNA): DNA version of mature mRNA, used in cloning to avoid non-coding introns.
• Plasmid Vector: Circular DNA used for inserting genetic information, replicates independently of host DNA.
• Insert and Vector: Insert is the genetic sequence; the vector is the plasmid carrying the sequence.

Plasmid Components

• Promoter sequence for transcription and translation, crucial in protein expression.
• Origin of replication to allow the plasmid to replicate independently in host cells.
• Selection marker like an antibiotic resistance gene to identify cells that have taken up the plasmid.

Molecular Cloning Techniques

Restriction Cloning

• Utilizes restriction enzymes (site-specific endonucleases) to cut DNA at specific sequences, creating sticky or blunt ends.
• Sticky ends allow for easier ligation of DNA fragments.
• Process typically involves: cutting vector and insert with the same enzyme → purify desired DNA fragments → ligate fragments

PCR-Based Methods

• Polymerase Chain Reaction (PCR): Technique to amplify specific DNA sequences.
• Primers: Short DNA sequences that start DNA synthesis; can be designed to include restriction sites.
• Phosphorylation considerations: Necessary for ligation; some DNA ends need phosphate groups.

Specific Methods in Detail

Sequence and Ligation Independent Cloning (SLIC)

• Utilizes PCR to amplify insert and vector with overlapping regions.
• T4 polymerase chews back DNA to create overhangs, allowing for homologous recombination in bacteria.
• Avoids pre-ligation, recombination fills gaps in the bacterial cells.

Gibson Assembly

• Similar to SLIC but uses different enzymes for exonuclease, polymerase, and ligase functions.
• More costly, but effective for cloning.

Golden Gate Cloning

• Uses Type IIs restriction enzymes that cut outside their recognition sequence, allowing seamless assembly.

Verification Methods

• Colony PCR or Analytical Digest to check for correct insert size.
• DNA sequencing to confirm the correct sequence of the insert.

Transformation into Bacteria

• Transformation: Process of introducing plasmid DNA into bacteria.
• Transfection: Similar process in non-bacterial cells.
• Transduction: Viral introduction of DNA into cells.
• Methods: Heat shock, electroporation.

Controls and Screening

• Positive controls: Ensure competent cells and transformation efficiency.
• Negative controls: Confirm no parental plasmid or self-ligation.
• Screening methods: Blue/white screening, antibiotic resistance.

Summary

• Molecular cloning techniques are diverse but follow similar principles of inserting and verifying genetic sequences.
• Proper verification and controls are crucial for successful cloning.