Rucete ✏ Campbell Biology In a Nutshell
Unit 3 GENETICS — Concept 20.1 DNA Sequencing and DNA Cloning Are Valuable Tools for Genetic Engineering and Biological Inquiry
DNA sequencing and cloning have transformed biological research, allowing scientists to precisely study and manipulate genetic material. These technologies are foundational for genetic engineering, biotechnology, medicine, agriculture, and numerous fields of biology.
DNA Sequencing Techniques
- DNA sequencing determines the precise order of nucleotides in DNA molecules.
- Dideoxy sequencing (Sanger sequencing): Developed by Frederick Sanger, useful for small-scale projects.
- Next-generation sequencing (high-throughput sequencing):
- Rapid and inexpensive.
- DNA fragments (~300 bases) are sequenced simultaneously.
- Enables sequencing of billions of bases in hours.
- Third-generation sequencing (e.g., nanopore):
- Sequencing long DNA molecules directly.
- Detects bases by electrical signals as DNA passes through pores.
- Portable and cost-effective (e.g., handheld nanopore devices).
DNA Cloning
DNA cloning produces multiple identical copies of specific DNA segments. It is essential in genetic research and biotech applications.
- Commonly uses bacterial plasmids:
- Circular DNA naturally found in bacteria.
- Engineered to carry foreign DNA = recombinant DNA.
- Gene cloning:
- Foreign DNA is inserted into plasmids (vectors).
- Bacteria replicate, copying the plasmid and gene.
Restriction Enzymes and Recombinant DNA
- Restriction enzymes (endonucleases):
- Cut DNA at specific sequences (restriction sites).
- Produce sticky ends that can base-pair with matching DNA.
- DNA ligase: Joins DNA fragments permanently, sealing recombinant DNA.
- Gel electrophoresis: Separates DNA fragments by size and confirms cloning success.
Polymerase Chain Reaction (PCR)
- Rapidly amplifies DNA sequences in vitro.
- Involves repeating:
- Denaturation: DNA strands separate.
- Annealing: Primers bind to target sequences.
- Extension: DNA polymerase builds new strands.
- Billions of copies from trace DNA sources.
- Used in forensics, diagnostics, evolutionary studies, cloning.
Expressing Cloned Genes
- Expression vectors: Allow genes to be transcribed and translated in host cells (e.g., bacteria).
- Issues with eukaryotic gene expression in bacteria: different regulatory elements and introns.
- Solution: Use complementary DNA (cDNA), synthesized from mRNA, which lacks introns.
- Other hosts: yeasts and cultured animal cells (handle protein modification).
Practical and Research Applications
- Cloned genes can:
- Produce proteins (e.g., insulin, HGH).
- Engineer pest-resistant crops (GMO).
- Investigate gene function and expression.
Cross-Species Gene Expression
- Many genes work across species (e.g., Pax-6 gene in eye development), showing evolutionary conservation.
In a Nutshell
DNA sequencing and cloning are revolutionary tools for analyzing and manipulating genes. With PCR, restriction enzymes, and expression systems, they support applications in medicine, agriculture, and basic science. These technologies have unlocked the blueprint of life.