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?
Sign in to unlock this chapter
Biology chapters 1–3 are free. Sign up to unlock every chapter.