U01.01.043 Molecular cloning

Molecular cloning is a cornerstone technique in molecular biology that involves creating recombinant DNA molecules and propagating them in bacterial hosts. This method allows the amplification and study of specific DNA sequences.

Key Steps in Molecular Cloning

1. Isolation of Eukaryotic mRNA

Source: Isolate mRNA of interest from eukaryotic cells.
Why: mRNA represents the coding regions of genes, excluding introns, as it has undergone post-transcriptional processing.

2. Synthesis of Complementary DNA (cDNA)

Enzyme Used: Reverse transcriptase (an RNA-dependent DNA polymerase).
Process: Convert mRNA into cDNA.
Outcome: cDNA is intron-free, making it ideal for bacterial expression systems.

3. Insertion of cDNA into Plasmids

Plasmids: Circular DNA molecules used as vectors.
Enhancements: Plasmids are engineered to include antibiotic resistance genes for selection.
Procedure: Insert cDNA into plasmids using restriction enzymes and DNA ligase.

4. Transformation into Bacteria

Bacterial Host: E. coli is commonly used.
Process: Introduce recombinant plasmids into bacterial cells via transformation (e.g., heat shock or electroporation).

5. Selection and Cloning

Antibiotic Medium: Plate bacteria onto an antibiotic-containing medium.
Selection: Only bacteria containing the recombinant plasmid survive and proliferate.
Cloning: The bacteria replicate, producing multiple copies of the cDNA.

Summary Table of Steps in Molecular Cloning

Step	Key Action	Outcome
Isolation of mRNA	Extract eukaryotic mRNA	Obtain processed, coding RNA of interest
cDNA Synthesis	Use reverse transcriptase to make cDNA	Generate intron-free DNA
Plasmid Insertion	Ligate cDNA into bacterial plasmids	Create recombinant plasmids
Transformation	Introduce plasmid into bacterial cells	Transform bacteria with recombinant DNA
Selection & Cloning	Grow bacteria on antibiotic medium	Amplify cloned cDNA

Points to Remember

Reverse Transcriptase: Key enzyme for converting RNA to DNA.
Introns: Bacteria cannot process introns; using cDNA avoids this issue.
Plasmid Selection: Antibiotic resistance genes ensure that only transformed bacteria grow.
High Efficiency: Transformation methods must maximize plasmid uptake.

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