DEEP WORK

INTRODUCTIONReplication copies the entire set of genomic DNA so that the cell can divide in two. During replication, the entire genome must be uncoiled and copied exactly. This elegant process occurs extremely fast in E. coli, where DNA polymerase copies about 1000 nucleotides per second. Although the process is slower in eukaryotes, DNA polymerase still copies 50 nucleotides per second. Many bi..

RECOMBINEERING INCREASES THE SPEED OF GENE CLONINGAssembling new DNA vectors with different genes of interest can become difficult when the gene is long, since it can be hard to identify unique restriction enzymes compatible with a polylinker that do not cut within the gene. Large recombinant DNA vectors can be created using homologous recombination, a process called recombineering (Fig. 3.25). ..

FEATURES OF EXPRESSION VECTORSBecause foreign protein can be toxic to E. coli, especially if made in large amounts, the promoter used to express the foreign gene is critical.  critical 대단히 중요한 If too much foreign protein is made, the host cell may die. To control protein production, expression vectors have promoters with on/off switches; therefore, the host cell is allowed to grow and then after..

EUKARYOTIC EXPRESSION LIBRARIESIn expression libraries, the vector has sequences required for transcription and translation of the insert. This means that the insert DNA is expressed as RNA and then translated into a protein. An expression library, in essence, generates a protein from every cloned insert, whether it is a real gene or not.  When eukaryotic DNA is studied, expression libraries are..

SCREENING THE LIBRARY OF GENES BY HYBRIDIZATIONOnce the library is assembled, researchers often want to identify a particular gene or segment of DNA within the library. Sometimes the gene of interest is similar to one from another organism. Sometimes the gene of interest contains a particular sequence. For example, many enzymes use ATP to provide energy. Enzymes that bind ATP share a common sign..

GETTING CLONED GENES INTO BACTERIA BY TRANSFORMATIONOnce the gene of interest is cloned into a vector, the construct can be put back into a bacterial cell through a process called transformation (Fig. 3.18) (see Box 3.2).FIGURE 3.18 Transformation Bacterial cells are able to take up DNA such as recombinant plasmids by incubation with metal ions such as Ca++ on ice. This destabilizes the bacteria..

SPECIFIC TYPES OF CLONING VECTORS Because E. coli is the main host organism used for manipulating DNA, most vectors are based on plasmids or viruses that can survive in E. coli or similar bacteria. Most vectors have bacterial origins of replication and antibiotic resistance genes.  The polylinker or multiple cloning site is usually placed between prokaryotic promoter and terminator sequences (Fi..

GENERAL PROPERTIES OF CLONING VECTORS Cloning vectors are specialized plasmids (or other genetic elements) that will hold any piece of foreign DNA for further study or manipulation. The numbers and types of plasmids available for cloning have grown. genetic manipulation 유전자 조작  In addition, other DNA elements are now used, including viruses and artificial chromosomes.Once a fragment of DNA has b..

METHODS OF DETECTION FOR NUCLEIC ACIDS Recombinant DNA methodologies require the ability to detect DNA. One of the easiest ways to detect the amount of DNA or RNA in solution is to measure the absorbance of ultraviolet light at 260 nm (Fig. 3.4). DNA absorbs ultraviolet light because of the ring structures in the bases. Single-stranded RNA and free nucleotides also absorb ultraviolet light. In f..

RESTRICTION ENZYMES CUT DNA; LIGASE JOINS DNAThe ability to isolate, separate, and visualize DNA fragments would be useless unless some method was available to cut the DNA into fragments of different sizes. Luckily, naturally occurring restriction enzymes or restriction endonucleases are the key to making DNA fragments. visualize 상상하다endonucleases  ((DNA 혹은 RNA의 사슬을 분해하여 불연속화(不連續化)시키는 효소)) These..