Molecular diagnostics : fundamentals, methods, & clinical applications


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Lederberg and Tatum proved that genetic exchange between organisms was not restricted to the sexually reproducing molds. Bacteria with double mutations that required exogenous methionine and biotin met and bio or threonine and leucine thr and leu can- not grow on a medium without addition of these nutrients minimal medium. When these strains are mixed together, however, growth occurs. The F factor was subsequently shown to be an extra- chromosomal circle of double-stranded DNA carrying the genes coding for construction of the mating bridge.

Just as animal and plant viruses infect eukaryotic cells, bacterial viruses, or bacteriophages, infect bacterial cells.

Molecular Diagnostics: Fundamentals, Methods, & Clinical Applications

The structure of bacteriophage T4 is one example of the specialized protein coats that enable these viruses to insert their DNA through the cell wall into the bacter- ial cell Fig. The experiment showed that viral protein remained outside of the cell while viral DNA entered the cell. Furthermore, 32 P- labeled DNA could be detected in new viruses generated in the transduction process Fig.

Methods soon developed using bacteriophages to move genetic infor- mation between bacteria by growing the phage on one strain of bacteria and then infecting a second strain with those viruses.

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Transduction is also useful in determining gene order. Griffith was inves- tigating virulence in Diplococcus now known as Streptococcus pneumoniae. He had two strains of the bacteria: one with a rough colony type that was avirulent and one with a smooth colony type that was virulent. Griffith intended to use these strains to develop a protec- tive vaccine Fig. It has specialized structures.

Radioactive DNA, however, does enter right and is passed to subsequent generations of viruses. Surprisingly, when he mixed killed smooth-type and live rough-type bacteria, virulence returned. Furthermore, he could recover live smooth-type bacteria from the dead mice. What Griffith had observed was the transfer of DNA from one organism to another without the protection of a conjugative bridge or a viral coat. Fifteen years later, Oswald T. Avery, Colin MacLeod, and M. Protease and ribonuclease treatment, which degraded protein and RNA, respectively, did not affect the transformation phenomenon that Griffith had demon- strated earlier.

Treatment with deoxyribonuclease, which degrades DNA, however, prevented transformation.

Plasmids DNA helices can assume both linear and circular forms. Most bacterial chromosomes are in circular form. Chromosomes in higher organisms, such as fungi, plants, and animals, are mostly linear. The ends of linear chro- mosomes are protected by specialized structures called telomeres. A cell can contain, in addition to its own chro- mosome complement, extrachromosomal entities, or plasmids Fig. Most plasmids are double-stranded circles, —, bp 2— kilobase pairs in size. Just as chromosomes do, plasmids carry genetic informa- tion.

Due to their size and effect on the host cell, plas- mids can carry only a limited amount of information. The plasmid DNA duplex is compacted, or supercoiled.

Outlines & Highlights for Molecular Diagnostics: Fundamentals, Methods, & Clinical Applications

Breaking one strand of the plasmid duplex, or nicking, will relax the supercoil Fig. Different physical states of the plasmid DNA can be resolved by distinct migration characteristics during gel electrophoresis. Plasmids were discovered to be the source of resistant phenotypes in multidrug-resistant bacteria. Only destruction of DNA prevented transformation. Advanced Concepts Investigators performing early transformation stud- ies observed the transfer of broken chromosomal DNA from one population of bacterial cells to another.

Naked DNA transferred in this way, how- ever, is a very inefficient source for transformation. Unprotected DNA is subject to physical shearing as well as chemical degradation from naturally occur- ring nucleases, especially on the broken ends of the DNA molecules. Natural transformations are much more efficient, because the transforming DNA is in circular form.

R factors carry resistance to common antibiotics such as chloramphenicol, tetracy- cline, ampicillin, and streptomycin. Another class of plasmid, colicinogenic factors, carries resistance to bac- teriocins, toxic proteins manufactured by bacteria.

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Plasmids can replicate in the host cell but cannot survive outside of the cell as viruses do. The acquisition of the resistance genes from host chromosomes of unknown bacteria is the presumed origin of these resistance fac- tors. Simultane- ous introduction of R factors into a single cell can result in recombination between them, producing a new, recom- binant plasmid with a new combination of resistance genes. Large plas- mids include the F factor and some of the R plasmids. Large plasmids carry genes for their own transfer and propagation and are self-transmissible.

Large plasmids occur in small numbers, one or two copies per chromo- some equivalent. Small plasmids are more numerous in the cell, about 20 copies per chromosomal equivalent; however, they do not carry genes directing their mainte- nance. They rely on high numbers for distribution into daughter cells at cell division or uptake by host cells in transformation. Compared with fragments of DNA, plasmids are more efficient vehicles for the transfer of genes from one cell to another.

Upon cell lysis, supercoiled plasmids can enter other cells more efficiently. Examination of the fractions of the higher density DNA revealed small circular species. These circles were absent from drug-sensitive bacteria.

As will be described in later chapters, plasmids play a key role in the development of procedures used in molecular analysis. What is the function of DNA in the cell? Compare the structure of the nitrogen bases. How do purines and pyrimidines differ? Which of the ribose carbons participate in the phos- phodiester bond?

Which of the ribose carbons carries the nitrogen base? Why does DNA polymerase require a primer? Restriction Enzyme Analysis 1.

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A plasmid was digested with the enzyme, HpaII. On agarose gel electrophoresis, you observe three bands, , , and bp. How many HpaII sites are present in this plasmid? What are the distances between each site? What is the size of the plasmid? Draw a picture of the plasmid with the Hpa II sites. A second cut of the plasmid with BamH1 yields two pieces, 80 and x bp. How many BamH1 sites are in the plasmid?

What is x in base pairs bp?

Molecular diagnostics : fundamentals, methods, & clinical applications Molecular diagnostics : fundamentals, methods, & clinical applications
Molecular diagnostics : fundamentals, methods, & clinical applications Molecular diagnostics : fundamentals, methods, & clinical applications
Molecular diagnostics : fundamentals, methods, & clinical applications Molecular diagnostics : fundamentals, methods, & clinical applications
Molecular diagnostics : fundamentals, methods, & clinical applications Molecular diagnostics : fundamentals, methods, & clinical applications
Molecular diagnostics : fundamentals, methods, & clinical applications Molecular diagnostics : fundamentals, methods, & clinical applications
Molecular diagnostics : fundamentals, methods, & clinical applications Molecular diagnostics : fundamentals, methods, & clinical applications
Molecular diagnostics : fundamentals, methods, & clinical applications Molecular diagnostics : fundamentals, methods, & clinical applications
Molecular diagnostics : fundamentals, methods, & clinical applications Molecular diagnostics : fundamentals, methods, & clinical applications

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