PCB 3063 Revised Exam 4 2025

Recombinant DNA - -joining of DNA molecules; produced by artificially joining DNA from different biological sources not found together in nature Cloning - -the process of making many copies of a specific DNA sequence Types of cloning - -PCR and cloning with a vector through recombination Restriction enzymes - -produced by bacteria as a defense mechanism against bacteriophages; DNA-cutting enzymes Palindrome sequences - -targeted by restriction enzymes, symmetry exhibited by recognition sequence and allow enzymes to cut both strands Sticky ends - -Single stranded ends of DNA left after cutting with enzymes Blunt ends - -fragment ends of a DNA molecule that are fully base paired, resulting from cleavage by a restriction enzyme DNA ligase - -seals the phosphodiester backbone to create intact DNA Vectors - -carrier DNA molecules; can replicate in host cells independent of host cell chromosome; usually have many restriction enzymes Selectable gene marker - -distinguish host cells that have taken them up from those that have not; usually an antibiotic resistance gene Plasmids - -extrachromosomal double-stranded DNA molecules and can be genetically modified Transformation - -process in which plasmids are introduced into bacteria 2 main techniques of transformation - - 1. Use calcium ions and brief heat shock to force cells to take up DNA 2. Electroporation Electroporation - -a brief but high intensity pulse of electricity to move DNA into bacterial cells Selectable marker genes - -genes that provide resistance to antibiotics Blue-white selection - -Used to identify cells containing recombinant And no recombinant DNA; plasmid contains lacz gene, which encodes B-galactosidase PCB 3063 PCB 3063 Blue-white screening mechanism - -agar plates contain X-gal, when X-gal is cleaved by enzyme it turns blue Cell type that turns blue from blue-white mechanism - -bacterial cells with functional lacz gene carrying no recombinant plasmid Cell type that turns white from blue-white mechanism - -bacterial cells with recombinant plasmid DNA libraries - -represent collection of cloned DNA fragments (genomic and complementary) Genomic library - -complete collection of cloned DNA fragments from an organism Complementary DNA (cdna) library - -Contains complementary DNA copies made from mrnas present in cell population; Represents genes active transcriptionally at the time cells were collected for mrna isolation Cdna library is constructed by - -isolating mrna from cells, synthesizing complementary DNA using reverse transcriptase, cloning cdna molecules into vector Polymerase chain reaction - -rapid method of DNA cloning that eliminates the need to host cells for cloning PCR steps - -1. Denaturation 2. Primer annealing 3. Extension (steps repeated over and over) PCR primers - -short, single stranded, one complementary to each end What do primers do - -anneal to denature DNA How are complementary strands synthesized? - -heatstable DNA polymerase Limitations of PCR - -contamination from other sources (ANY DNA that the primers bind to will be amplified), high error rate, some info is required Applications of PCR - -diagnostic tests, screens for mutations, widely used in genetics and molecular biology Reverse transcription PCR - -generate cdna from RNA, then converted to DNA Quantitative real-time PCR - -allows researchers to quantify amplification reactions as they occur in real time PCB 3063 PCB 3063 Restriction maps - -show the lengths of DNA fragments between restriction sites in a strand of DNA Southern blot - -used to characterize size and identify of DNA fragments; separate, transfer, hybridization Northern blot analysis - -uses probes to detect mrna, used to determine whether gene is actively being expressed in given cell or tissue Western blot - -used to analyze proteins Most common method of DNA sequencing - -dideoxynucleotide chain-termination sequencing (Sanger sequencing) Dideoxynucleotide chain-termination sequencing (Sanger sequencing) - -most common, begins with primer to bind with strand to be sequenced, dideoxynucleotide (ddntp) with hydrogen 3' end instead of OH Next generation sequencing - -identify individual bases as they are added to the growing strand, dramatically improved the speed of sequencing Gene targeting - -concept is to manipulate specific allele, locus, or base sequence and learn its function Gene knockout/mutation - -concept is to disrupt or eliminate specific gene/genes and determine how this affects phenotype (reverse genetics) Result of target vectors introduces into cells - -chimeras- containing some mutant and some WT cells Cre-lox system - -uses the enzyme Cre-recombinase to delete a sequence flanked by lox sites (specific target sequence for the enzyme) Transgenic animals- knock-in animals - -Express or overexpress particular gene of interest (transgene) Vector with transgene undergoes homologous recombination into host genome Vector with transgene can also be put into ES cells and injected into embryos Allows for study of effects on appearance and function in mice CRISPR-Cas9 system - -guide RNA designed to bind to specific sequence, Cas9 detects the guide RNA and cuts DNA, insertion Genomics purpose - -allows sequencing and analysis of entire genomes Genome - -complete set of DNA in a single cell of an organism PCB 3063 PCB 3063 Genomics - -the study of genomes Whole-genome sequencing (shotgun cloning) - -The most widely used strategy for sequencing and assembling an entire genome Whole-genome sequencing method - -1. Genomic DNA is cut into fragments and contigs made 2. Entire chromosome is assembled by computer program 3. Fragments are aligned based on identical DNA sequences Bioinformatics - -uses computer-based approaches to organize, share, and analyze large datasets related to gene structure, gene sequence and expression, and protein structure and function Genbank - -NCBI database of sequences, each with its own accession number Annotation - -relies on bioinformatics, identifies genes, their regulatory sequences, and their functions BLAST (basic local alignment search) - -software application using similarity scores to identify portion of DNA, E-value Structural genomics - -analyzing nucleotide sequences to identify genes and sequences such as gene-regulatory elements Functional genomic - -study of gene functions based on resulting rnas and possible proteins they encode, also identifies gene regulatory elements, their functions, and interaction, comparisons are made with known sequences in other individual/species using established databases Homologous genes - -genes that are evolutionarily related and often have similar functions, similarity searched can identify homologous genes Orthologs - -genes from different species thought to have descended from common ancestor Paralogs - -homologous genes in same spe