Recombinant DNA and Biotechnology
High-Yield Summary
- Gene cloning: restriction enzymes cut DNA at palindromic recognition sites, creating sticky ends; fragment inserts into a plasmid vector forming recombinant DNA; DNA ligase seals it; bacteria replicate/express it (e.g. bulk insulin production).
- Genomic library = entire genomic DNA (exons + introns), comprehensive. cDNA library (expression library) = reverse-transcribed from mRNA, exons only — reflects actively expressed genes.
- Hybridization (pairing of complementary DNA-DNA or DNA-RNA strands) underlies PCR, gel electrophoresis, Southern blotting, and sequencing.
- PCR cycles: denaturation (~95°C, separates strands) → annealing (~55°C, primers bind) → extension (72°C, Taq polymerase synthesizes) — exponential amplification.
- Gel electrophoresis separates DNA by size (negatively charged DNA migrates toward positive electrode; smaller fragments move faster). Southern blot adds a labeled complementary probe after transfer to a membrane, to detect/quantify a specific sequence.
- Dideoxy sequencing: ddNTPs lack a 3'-OH, so DNA polymerase can't extend past one — chain-terminating fragments of varying length, separated by electrophoresis, read the sequence.
- Applications: gene therapy (functional gene delivered via a modified viral vector) and transgenic (foreign transgene added) vs. knockout (gene deleted) mice — both require safety/ethics oversight (e.g. insertional mutagenesis risk).
Key Terms
- Restriction enzyme
- Cuts DNA at specific, often palindromic, recognition sites, producing sticky ends.
- Recombinant DNA
- Hybrid DNA molecule combining sequences from two different sources (e.g. gene + plasmid vector).
- cDNA library
- Collection of DNA reverse-transcribed from mRNA — exons only; reflects actively expressed genes.
- Taq polymerase
- Heat-stable DNA polymerase used in PCR to survive repeated high-temperature denaturation steps.
- Southern blot
- Technique detecting a specific DNA sequence via gel transfer + labeled complementary probe hybridization.
- Dideoxyribonucleotide (ddNTP)
- Chain-terminating nucleotide lacking a 3'-OH, used in DNA sequencing.
Genomic Library vs. cDNA Library
| Feature | Genomic vs. cDNA Library |
|---|---|
| Source | Entire genomic DNA / mRNA, reverse-transcribed |
| Contents | Exons + introns / Exons only |
| Best for | Comprehensive DNA coverage / Studying actively expressed genes |
PCR Cycle
- 1Denaturation (~95°C): heat separates the double-stranded DNA into two single strands.
- 2Annealing (~55°C): short primers hybridize to their complementary sequences on each strand.
- 3Extension (72°C): Taq polymerase synthesizes new complementary DNA from each primer.
- 4Repeat the three steps for many cycles — DNA doubles each cycle, producing exponential amplification.
Worked Example — Cloning the Insulin Gene
- 1Start with a plasmid and the gene of interest (insulin gene).
- 2Digest both with the same restriction enzyme (e.g. EcoRI), creating complementary sticky ends.
- 3DNA ligase seals the fragment into the plasmid backbone, forming recombinant DNA.
- 4Introduce the plasmid into bacteria — it replicates and/or expresses the gene to produce insulin protein.
Common MCAT Trap
- A genomic library includes introns and non-coding regions — it is NOT the right tool for studying gene expression. Use a cDNA (expression) library for that.
- PCR temperatures are a common numeric-recall question: ~95°C denature, ~55°C anneal, 72°C extend — don't swap annealing and extension temperatures.
- ddNTPs stop chain extension because they lack a 3'-OH (not because they lack a base or phosphate) — DNA polymerase needs that hydroxyl to add the next nucleotide.
Quick Recall
What feature of restriction enzyme recognition sites allows sticky-end pairing?
Why is a cDNA library called an expression library?
What three temperature steps make up one PCR cycle?
Why do smaller DNA fragments migrate farther in gel electrophoresis?