Genome, Genomics and DNA Cloning

Genome and Genomics

• The genome is the complete haploid genetic complement of an organism.
• Genomics is the study of DNA on a cellular scale.
• Genomics contributes to systems biology, the study of biochemistry on whole cells and organisms.

Genes and DNA Cloning

• A clone is an identical copy.
• DNA cloning selectively amplifies a gene or DNA segment for study and utilization.
• Recombinant DNA technology, or genetic engineering, includes methods for DNA cloning and related tasks.

Process of DNA Cloning

• DNA cloning involves obtaining a DNA segment, selecting an autonomous replication molecule, joining the fragments covalently, and moving recombinant DNA to a host.
• Host cells containing the recombinant DNA must be selected or identified.
• Cloning vectors are small DNAs capable of autonomous replication.
• Recombinant DNAs are composite DNA molecules with covalently linked segments from two or more sources.
• E. coli cloning offers advantages like well-understood DNA metabolism and naturally occurring cloning vectors like plasmids.
• E. coli cloning has techniques for moving DNA from one bacterial cell to another.

Restriction Endonucleases

• Restriction endonucleases, or restriction enzymes, recognize and cleave DNA at specific sequences, called recognition sequences or restriction sites.
• Methylases catalyze host DNA methylation, protecting it from digestion by the host cell's restriction endonucleases.
• A restriction-modification system encompasses the restriction endonuclease and its corresponding methylase.
• Type II restriction endonucleases are simpler than Types I and III, require no ATP, and catalyze hydrolytic cleavage of DNA phosphodiester bonds within the recognition sequence.
• Restriction endonucleases can produce sticky or blunt ends.
• Sticky ends are unpaired bases on the ends making staggered cuts that can base pair with each of their complementary sticky ends.
• Blunt ends are ends with no unpaired bases on the ends due to endonucleases making straight cuts.

Restriction Enzymes and DNA Ligases

• DNA ligases join DNA fragments to cloning vectors.
• Including restriction endonuclease cleavage sites facilitates the subsequent cloning of amplified DNA.
• Cleavage of PCR-amplified DNA creates sticky ends used to ligate the amplified DNA to a cloning vector.
• Linkers are synthetic DNA fragments used to bridge ligated ends.
• Multiple cloning sites (MCS) are inserted DNA fragments with multiple recognition sequences for restriction endonucleases, useful for inserting additional DNA later.

Plasmids and Vectors

• Plasmids are circular DNA molecules that replicate independently from the host chromosome.
• Plasmids range from 5,000 to 400,000 bp and often have a symbiotic role in the cell.
• A constructed E. coli plasmid, pBR322, has key features like an origin of replication, resistance genes, and recognition sequences for restriction endonucleases.
• Shuttle vectors are plasmids propagated in two or more species and incorporate multiple replication origins or other elements.
• Bacterial transformation is a lab process that introduces small plasmids into bacterial cells using heat shock treatment.
• Electroporation is a lab process that introduces small plasmids into bacterial cells through high-voltage pulses, transiently rendering the bacterial membrane permeable.
• Selectable markers identify cells that have taken up plasmid DNA, permitting growth or killing cells under specified conditions.
• Screenable markers are genes encoding proteins that produce a colored or fluorescent molecule.

Expressing Cloned Genes

• Expression vectors are cloning vectors with transcription and translation signals for regulated expression of a cloned gene.
• Purified proteins can elucidate protein function, study reaction mechanisms, generate antibodies, reconstitute complex activities, and examine protein-binding partners.
• Virtually any organism can host protein expression from a different species, including bacteria and yeast.
• Bacterial hosts are commonly used due to well-understood regulatory sequences, high expression levels, easy storage, and efficient transformation.
• However, some heterologous proteins may not fold correctly and eukaryotic proteins may aggregate into insoluble precipitates, known as "inclusion bodies".
• The Saccharomyces cerevisiae is a typically utilized and well-understood eukaryotic organism.
• Principles of protein expression in yeast are similar to bacteria: Cloned genes require appropriate promoters, while gene expression is controlled by selecting an appropriate medium.
• Mammalian cells in culture use engineered mammalian viruses as vectors.
• Protein changes and Alterations: Site-directed mutagenesis can be used to replace specific amino acids.
• Alterations to Cloned Genes product altered proteins. Site-directed mutagenesis is used to individually replace specific amino acids. Oligonucleotide-directed mutagenesis is also used to create a specific DNA sequence change.

Affinity Purification Tags

• Terminal tags are peptides or proteins that bind a simple, stable ligand with high affinity and specificity.
  • Used to fuse to genes encoding target proteins.
  • Permits purification by affinity chromatography.

PCR

• Reverse transcriptase PCR (RT-PCR) uses reverse transcriptase to generate DNA from an RNA template, followed by standard PCR.
• Quantitative PCR (qPCR) or real-time PCR is used to estimate relative copy numbers of particular sequences in a sample
• A DNA library contains a collection of DNA clones for gene discovery and determining gene or protein function.
• Combinatorial or gene libraries focus on sequence variations within a gene.
• Complementary DNAs (cDNAs) are double-stranded DNA fragments formed from mRNA templates. They depend on reverse transcriptases.
• Clicker Question: Restriction endonucleases recognize and cleave DNA at specific sequences.
• Clicker Question: The operator of a typical E. coli expression vector permits regulation by a repressor that binds to it.
• Clicker Question: Tags for affinity purification may affect protein folding.

Protein Function

• Phenotypic function describes effects on the entire organism.
• Cellular function describes interactions at the cellular level.
• Molecular function describes precise biochemical activity.
• Transcriptome is the entire complement of transcribed RNAs, and its study is transcriptomics.
• Proteome is the entire complement of proteins, and its study is proteomics.
• Comparative genomics assigns gene functions using genome databases, with BLAST algorithms facilitating rapid searches.
• Genome annotation converts the sequence of residues into useful information about the location and function of genes and other critical sequences.
• Orthologs are genes in different species with a clear sequence and functional relationship.
• Paralogs are similarly related genes within a single species.
• Synteny is the conserved gene order, it provides additional evidence for an orthologous relationship between genes at identical locations within the related segments.
• Sequence-associated structural motifs help define the molecular function.

Methods to determine protein function

• Location analysis of protein and when: Proteins involved in reactions/processes must be present in specific location and moment. Techniques: RNA-Seq/transcriptomics, cell proteomes/mass spectrometry, fusion proteins/immunofluorescence.
• Mass spectrometry accurately catalogs and quantifies proteins providing info about modified proteins complementing RNA-Seq.
• Green fluorescent protein (GFP), a jellyfish protein, is a useful location marker that is of other colors and characteristics.
• Immunofluorescence is an alternative approach for visualizing cellular endogenous protein.
• Epitope tags are bound to a short and attached protein sequence by an antibody tightly.
• Techniques: Purification of protein complexes and yeast two-hybrid analysis
• CRISPR sequences regularly repeating sequences that use Cas protein for protection and immune defense.
• The CRISPR-Cas protein complex contains transcribed viral sequences that are cleaved that activates CRISPR RNA and has at least +1 Cas proteins.
• The complex destroys invading bacteriophage DNA by the Cas protein nuclease activities.
• CRISPR Technology consist of Cas protein and can alter guide sequence and target genomic sequences.
• Clicker Question: Cloning vectors are not sometimes plasmids, small circular DNA molecules lacking an ori sequence.
• Clicker Question: Tags for affinity purification of tags may affect protein folding.