Molecular Biology Techniques Quiz

DNA Libraries and Screening for Recombinants

• A DNA library is a collection of DNA clones representing either the entire genome of an organism or a set of genes expressed in a specific cell type.
• Gene libraries are sets of cloned DNA fragments representing the genes of a specific organism.
• Isolating specific genes directly from a genome is impractical due to the large quantity of extraneous DNA.
• Genomic libraries are made by isolating total DNA, digesting it into suitable fragments, and cloning those fragments into appropriate vectors.
• Shotgun cloning is a strategy to clone all genes in an organism without targeting specific genes.
• Recombinant clones are screened to find those containing the desired gene.
• DNA is frequently partially digested to ensure that not every restriction site is cleaved in every DNA molecule.
• Overlapping fragments allow the reconstruction of the original DNA sequence.

Construction of Genomic Libraries

• Isolate Genomic DNA: Extract DNA from the organism of interest, ensuring high quality through methods like phenol-chloroform extraction and ethanol precipitation. Quantify and check purity using spectrophotometry or gel electrophoresis.
• Fragmentation: Digest DNA into manageable fragments using restriction enzymes or mechanical shearing (like sonication).
• Size Selection: Use agarose gel electrophoresis to select the DNA fragments of the desired size range, then extract using gel extraction methods.

Prepare the Vector

• Choose vectors appropriate for fragment sizes (plasmids, lambda phage vectors, BACs or YACs).
• Digest the vector using compatible restriction enzymes, opening it at the appropriate site.
• Dephosphorylate the vector to prevent self-ligation.

Ligate DNA Fragments into the Vector

• Mix the fragmented genomic DNA with the prepared vector in the presence of DNA ligase and ATP.
• DNA ligase joins the DNA fragments to the vector, creating recombinant DNA.

Transform Host Cells

• Introduce the recombinant DNA into a host organism (e.g., E. coli) using methods like heat shock, electroporation or in vitro packaging.

Plate the Transformants

• Spread the transformed cells on selective agar plates containing antibiotics or other markers to select for successful transformants.

Screen the Library

• Colony Hybridization: Use labeled probes to detect colonies containing specific DNA sequences.
• PCR-Based Screening: Amplify target sequences to identify desired clones.
• Store the genomic library as glycerol stocks at -80°C or as DNA libraries in appropriate buffers for long-term use.

Applications of Genomic Libraries

• Identifying genes and regulatory elements.
• Studying genetic disorders and mutations.
• Sequencing entire genomes.
• Producing recombinant protein or transgenic organisms

cDNA Cloning

• A cDNA library is a collection of DNA clones representing expressed genes in a specific cell or organism.
• cDNA is made from completely transcribed mRNA.
• cDNA libraries contain only expressed genes, lacking non-coding regions.
• They're useful for studying expressed genes and generating protein products.

Construction of cDNA Libraries

• Isolating mRNA: Isolate mRNA (most crucial step). All components should be super clean and free of chromosomal DNA, RNases and phenolics.
• Synthesis of 1st strand: Synthesis of cDNA using reverse transcriptase based on an mRNA template.
• Synthesis of 2nd strand: Synthesis of second cDNA strand using DNA polymerase.

Key Molecules in cDNA Construction

• Reverse transcriptase (RTase): Synthesizes the first cDNA strand using mRNA as a template.
• Oligo dT primers: Essential for mRNA purification.
• DNA Polymerases: Synthesize the second cDNA strand using the first strand as a template.
• S1 nuclease: Digests hairpin loops.

Total RNA and mRNA Isolation

• mRNA purity is crucial for cDNA synthesis success.
• Use DEPC-treated materials and bake glassware to prevent RNAse contamination.
• Grinding tissue in liquid nitrogen helps inhibit RNAse activity.

Purify the mRNA

• Use matrix columns containing oligo (dT) to isolate pure mRNA from total RNA.

cDNA Library Screening Methods

• Methods: Hybridization-based screening, PCR-based screening, and immunological screening.
• Probes: Pieces of DNA or RNA used to detect specific nucleic acid sequences via hybridization (binding of complementary nucleic acids).

Antibody Screening

• Identify translation products indirectly by raising antibodies specific to purified products.
• Screening procedure is similar to hybridization-based screening.

Screening of Libraries

• Probes can be labelled radioactively to detect hybridized nucleic acid via autoradiography.

• Probes vary in size from a few nucleotides to hundreds of kilobases.

• Initially, a probe may be double-stranded which must be single-stranded for screening purposes.