Definition: A single DNA molecule formed from two different sources via molecular cloning (a method of genetic engineering).
Importance:
Enabled isolation and manipulation of DNA, revolutionizing biotechnology.
Genes from slowly reproducing animals can now be introduced into rapidly-growing bacteria for expression.
Restriction Endonucleases
Function: Cut DNA at specific sites, facilitating the creation of recombinant DNA.
Origin: Evolved in bacteria as a defense mechanism against bacteriophage infections.
Key Points:
Can produce DNA fragments with sticky ends for easy joining.
Recognize specific DNA sequences known as restriction sites (usually palindromic and 4 to 12 bp long).
Gel Electrophoresis
Process: DNA fragments are separated by size in a porous agarose gel.
Mechanism:
DNA is negatively charged, migrating towards the positive pole when subjected to an electric field.
Smaller fragments move faster than larger ones.
Visualization: DNA is stained with fluorescent dyes for analysis.
DNA Ligase Functionality
Role: Joins two DNA fragments using ATP, creating stable DNA molecules.
Comparison: Same enzyme is responsible for joining Okazaki fragments during DNA replication.
Recombinant DNA Replication
Utilization in Host: E. coli is commonly used to replicate recombinant DNA.
Process: Involves ligating a DNA fragment of interest to a plasmid (vector) and introducing it to E. coli via transformation.
Outcome: Bacteria replicate the recombinant DNA in each generation.
cDNA and Libraries
Reverse Transcriptase: Converts mRNA into complementary DNA (cDNA) allowing analysis of processed mRNA sequences.
DNA Libraries: Collections of DNA or cDNA maintained in a host organism, facilitating replication and study. Includes:
Cloning vectors with replication sequences, selectable markers, and restriction sites.
cDNA Libraries: Constructed from mRNAs representing expressed genes at specific conditions or tissues, enabling comparisons among different cell types.
17.2 Amplifying DNA Using PCR
Definition: PCR mimics DNA replication to amplify specific DNA sequences.
Components: Requires DNA template, primers, DNA polymerase, and nucleotides (dNTPs).
Three Steps of PCR:
Denaturation: High temperature separates DNA strands.
Annealing: Primers attach to target sequences at lower temperature.
Elongation: Taq polymerase synthesizes new DNA strands.
Impact of PCR: Revolutionized DNA amplification beyond basic research, now used in forensics, environmental DNA analysis, and more.
Quantitative RT-PCR
Purpose: Measures mRNA levels using cDNA conversion and subsequent PCR amplification.
Technique: Real-time quantification of PCR products through fluorescent probes, allowing analysis of gene expression levels.
PCR in Diagnostic Tests
Application: Rapid identification of infectious agents using specific primers for target genes.
Challenges: RNA viruses require reverse transcription PCR (RT-PCR) due to the delicacy of RNA molecules.
17.3 Creating and Analyzing Genetic Variation
DNA Fingerprinting: Uses short tandem repeats (STRs) for individual identification; valuable in forensic science.
Point Mutations via PCR: Specific mutations can be engineered in DNA sequences using tailored PCR primers.
RNA Interference: Reduces specific gene products in cells by degrading complementary mRNA.
CRISPR/Cas9 Editing: A simple genome editing technique utilizing RNA-guided technology to cut and modify DNA sequences.
17.4 Constructing Transgenic Organisms
Transgenics: Organisms genetically altered to express genes from other species.
Knockout Mice: Genetically modified mice where a specific gene has been disrupted to study gene function.
“Knockin” Mice: Specific alterations made to normal alleles to study gene function.
17.5 Environmental Applications
Biofuels: Derived from biomass, offering renewable energy sources compared to fossil fuels.