Chapter 7: Techniques of Molecular Biology Flashcards
Molecular Techniques
Molecular techniques
Protein purification
- Size exclusion
- Ion exchange
- Affinity chromatography
Protein Analysis
- SDS page
- 2D Gel Electrophoresis
- Western Blotting
Forward and Reverse Genetics
Forward genetic determines the basis for a phenotype by working from the protein to the gene
Reverse Genetics is starting with the known gene and discovering the protein and its phenotype
The gene sequence deduced from the amino acid sequence of a protein is ambiguous due to the degeneracy of the codons
Biotechnology Foundations
Protein size, charge , and function were initially used to provide the basis for purification and separation
Beta-Globin Cloning
Was the first mammalian gene cloned. Beta-Globin was cloned using restriction enzymes, a plasmid, and a phage. Probe could be used to identify location in genome.
Dystrophin Cloning
Dystrophin is involved in muscle attachment. Subtractive cloning was performed.
- Sonicate the DNA from the patient.
- Digest normal DNA with enzyme MBO l
- Mix them
A clonable strand forms where both strands have the Mbo-cleaved ends
Southern blot
Used to analyze RNA using a DNA probe. A southern blot was used to create a human linkage map
Gene Mapping
Use different restriction sites across the genome and use it as markers. The limitations of gene mapping is the amount of time needed to analyze human genes
Genomic and cDNA Libraries
Gene libraries is the entire genome or protein-coding genes that can be sectioned or captured in bacteria or phage.
Gene libraries can be genomic or cDNA libraries which are screened with radio-labeled probes
A Genomic library captures introns, exons, and intergenic DNA. Genes can be transcribed, but E coli. does not translate due to intronic sequences. Genomic libraries can only be screened with DNA probe
A cDNA library represents the mRNA of a cell or tissue. Target sequence is better represented because it has less information. E coli. cells make the corresponding human proteins. cDNA libraries can be screened with DNA probes and antibody probes
Genome Sequencing
Chromosome walking involves using overlapped regions within genomic libraries to probe and re-screen to identify overlapping clones.
Shotgun sequencing involves breaking the genome into fragments for sequencing, and then aligning the results
Modern Molecular Biology
Genomics - The assessment of the entire genomes or organisms. Mapping and comparing genomes of individuals or species, studying genome structure, function, evolution, and SNPs
Proteomics - Studying proteins in the cell and their transcription, regulation, structures, and functions. Aims to identify the full set of proteins in a cell or tissue, and their modifications.
Systems Biology - Integrates strategies to study higher levels of biological organization through advanced analysis and modeling
Synthetic Biology - Artificial networks that mimic the features of natural pathways of gene control
Personal Genomics
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