Polymerase chain reaction, production determination

Krahmer, M. T. Johnson, Y. A. Walters, J. J. Fox, K. F. Fox, A. Nagpal, M. Electrospray quadrupole mass spectrometry analysis of model oligonucleotides and polymerase chain reaction products determination of base substitutions, nucleotide additions/deletions, and chemical modifications. Anal. Chem. 1999, 71, 2893-2900. 

M. Galloway and S.A. Soper, Contact conductivity detection of polymerase chain reaction products analyzed by reverse-phase ion pair microcapillary electrochromatography, Electrophoresis, 23 (2002) 3760-3768. M. Masar, M. Dankova, E. Olvecka, A. Stachurova, D. Kaniansky and B. Stanislawski, Determination of free sulfite in wine by zone electrophoresis with isotachophoresis sample pretreatment on a column-coupling chip, J. Chromatogr. A, 1026 (2004) 31-39. 

The plaque assay is desirable because it is very sensitive and only detects infectious viral particles. However, there are viral agents which cannot be supported by cell lines. In these cases other methods must be used. The polymerase chain reaction (PGR), which amplifies DNA or RNA from viral agents, can be used to detect the presence and quantity of viral agents. The amount of RNA or DNA target in the initial sample can be determined by competitive PGR where the quantity of amplified product is compared to a control PGR product where the initial amount of target is known. Quantification is also possible by an end-point dilution method similar to that used to determine a tissue culture infections dose. PGR methods can be very sensitive however. 

Quantitative polymerase chain reaction, also called real-time RT-PCR or QPCR, is a method which employs insertion of a signal, such as fluorescence or enzyme activity, into PCR products generated by RT-PCR to determine the amount of messenger RNA (mRNA) in a tissue accurately. 

A number of methods are available for analyzing tumor HER-2 status. The selection of the method depends on the target molecule to be detected. The target molecules are DNA mRNA, and protein (Fig. 12.2). HER-2 gene amplification can be detected by Southern blot (Press et al., 1994), slot blot (Naber et al., 1990), and dot blot assays (Descotes et al 1993), fluorescence in situ hybridization (FISH) (Persons et al., 1997), in situ hybridization (ISH) on isolated nuclei or tissue sections (Smith et al., 1994), and polymerase chain reaction (Gramlich et al., 1994). Assays to determine mRNA oveiexpiession include Northern blot (Slamon et al., 1989), Western blot (Press et al., 1994), slot blot (Naber et al., 1990) and ISH (Naber et al., 1990). Methods to assess HER-2/mcm protein product overexpression... 

The exception to limited technical coverage concerns the trend toward direct sequencing of plasmids and polymerase chain reaction (PCR) products. This is a reflection of the increasing need for ease and rapidity in sequence determination. However, many published protocols lack consistency. As discussed at greater length below, there are few or no (as yet) tried-and-true favorites. Thus, the techniques presented in this chapter comprise detailed protocols for only one fairly robust rapid isolation and... 

The polymerase chain reaction (PCR) is an ideal tool for the study of ancient DNA because it has the ability to amplify a small number of intact DNA molecules that exist in a complex mixture of large amounts of partially degraded and modified templates. Of crucial importance for the use of ancient DNA extracts is the extent to which the damage limits or inhibits the enzymatic reaction. An observation often made is that the maximum sizes of amplifiable products are reduced in old, damaged DNA compared to modem DNA extracts.14 This is also true for DNA from ancient bones, which seem in many cases to allow for longer amplifications than soft tissues. For example, we have determined the maximum size of amplifiable DNA from 3500-year-old moas found at a dry cave site in New Zealand and found that, whereas soft tissues allowed the amplification of pieces only up to 120 bp, bone extracts from the same individual yielded products of up to 380 bp. However, DNA extracted from a modem ratite bird easily allowed the amplification of pieces of over 1000 bp.15... 

The polymerase chain reaction (PCR), branched-DNA analyses, and related techniques are highly sensitive methods to quantify both viral RNA and provi-ral DNA. Proviral DNA is a measure of the number of cells infected, whereas viral RNA reflects production of virions. As with the measurement of viral products, one cannot easily determine whether the nucleic acids measured represent infectious viruses. A number of commercially available technologies, exist for the measurement of HIV nucleic acids, since this methodology is now the standard of care for AIDS patients. Laboratories performing their own quantitative analyses have generally used a modification of the quantitative competitive (QC)-PCR (66). 

Isolate genes for thermophilic enzymes by polymerase chain reaction (PCR) and subclone into expression vectors for production in mesophilic host Employ and analyze immobilization methods for optimal performance Determine enzyme kinetics separately and in combination to optimize hydrogen production... 

---