Molecular Cloning Basics

• DNA from any organism can be cloned using molecular cloning.
• Target DNA sources for cloning include genomic DNA, mRNA (converted to cDNA), previously cloned DNA, and synthetic sequences.
• Target DNA used in cloning can contain complete or partial protein coding sequences and regulatory sequences.

Restriction Enzymes (RE)

• Restriction enzymes cut DNA at specific palindromic sequences.
• Restriction enzymes produce sticky ends (better for cloning) or blunt ends (require more ligase).
• Bacteria use restriction enzymes as a defense against foreign DNA.
• Methylation protects bacterial DNA from cleavage by restriction enzymes.

Plasmid Cloning Vectors

• Plasmids are circular DNA molecules used for cloning.
• Plasmids feature an origin of replication (ori), an antibiotic resistance gene for selection, and a multiple cloning site (MCS) with restriction sites.
• An example of a plasmid is pUC19 (lacZ selection, ampicillin resistance).

Transformation of Bacteria

• Transformation methods include heat shock (cheap, easy) and electroporation (high efficiency, expensive).
• Selection of transformed cells is achieved through antibiotic selection and blue-white screening (lacZ disruption).

Cloning Eukaryotic Genes

• Eukaryotic genes have introns, so mRNA (introns removed) is needed for cloning.
• mRNA is converted to cDNA using reverse transcriptase.
• mRNA purification uses poly-A tail binding (oligo-dT beads).

Recombinational Cloning (Gateway Cloning)

• Recombinational cloning utilizes natural bacteriophage recombination instead of restriction enzymes.
• The ccdB gene acts as a selectable marker (toxic protein kills unwanted cells).

Genomic Libraries

• Genomic libraries are collections of DNA fragments representing an entire genome, generated by partial restriction digestion (100-200 kb fragments).
• Target genes can be identified through PCR (if the sequence is known), functional complementation (gene function tests), and colony immunoassay (antibody screening).

CRISPR-Cas9 Gene Editing

• CRISPR-Cas9 is a bacterial immune system adapted for gene editing.
• The steps of the CRISPR-Cas9 system are as follows: foreign DNA (spacer) incorporates into bacterial genome (CRISPR loci); CRISPR RNA (crRNA) binds to Cas9; and the Cas9-crRNA complex finds and cuts invading DNA.
• Single guide RNA (sgRNA) guides Cas9 to target DNA for gene editing.
• Cas9 cuts the DNA, enabling gene knockout or modification.

Polymerase Chain Reaction (PCR)

• PCR is used to amplify DNA with the following steps: denaturation (95°C) to separate DNA strands; annealing (55-65°C) for primers to bind to template; and extension (70°C) for DNA polymerase to extend strands.
• qPCR (Quantitative PCR) monitors amplification in real-time using fluorescence.
• TA Cloning uses Taq polymerase, which leaves overhangs for direct cloning.

DNA Sequencing Techniques

• Sanger Sequencing uses dideoxynucleotides (ddNTPs) to terminate DNA synthesis and is the oldest method, still used for small sequences.
• Next-Generation Sequencing (NGS) enables massively parallel sequencing (high throughput) and is used in whole-genome sequencing & metagenomics.

Genomics & Transcriptomics

• Shotgun sequencing was used in the Human Genome Project (2003), which took 13 years and cost $2.7 billion.
• RNA-Seq measures gene expression differences and is more accurate than microarrays (doesn’t require prior genome knowledge).

Proteomics

• 2D PAGE (Two-Dimensional Gel Electrophoresis) separates proteins based on charge (isoelectric focusing) and molecular weight
• The Yeast Two-Hybrid System studies interactions between proteins.
• Tandem Affinity Purification (TAP-Tagging) identifies protein complexes.

Metabolomics

• Metabolomics analyzes small molecules in cells.
• Mass Spectrometry (MS) & NMR (Nuclear Magnetic Resonance) are used for metabolite detection.
• Metabolomics can be used to find cancer biomarkers (detects unique metabolites in tumor cells) and to study plant stress response (terpenes emitted during heat stress).

Molecular cloning allows gene insertion and replication in bacteria.

• CRISPR-Cas9 enables precise gene editing.
• PCR allows for rapid DNA amplification, while NGS enables whole-genome sequencing.
• Proteomics & Metabolomics help understand protein function and metabolic pathways.