Genomic DNA template

The method of PCR allows selective amplification from a complex genome by enzymatic amplification in vitro. The double-stranded genomic DNA template is denatured by heating, and the temperature is then decreased to allow oligonucleotide primers to hybridize (anneal) to their complementary sequences on opposite strands of the template. The... 

Several different approaches to microarray construction have been developed, including oligonucleotide- or DNA-based arrays on glass slides or nylon membranes (10). Here, we describe a robust CGH protocol based on the use of a PCR-generated microarray (6). Detailed descriptions of genomic DNA template preparation, labeling, microarray hybridization, and data analysis are provided. 

Figure 4. Individuals of the same species yield indistinguishable PCR-RFLP handing patterns. Two different PCR amplifications of the rDNA of the same species give the similar banding patterns. Intraspecific variation is apparently low in the amplified region, (4A) A - Negative control in which amplification was performed with no genomic DNA template, Hinfl RFLPs of PCR products B,C - two different Cardiocladius obscurus individuals, D,E- two different Dicrotendipes fumidus individuals, and Lane SM -100 bp size marker. (4B) Rsal RFLPs of PCR products A,B- two different Cardiocladius obscurus individuals, C,D- two different Dicrotendipes fumidus individuals, and Lane SM - 100 bp size marker.

Virus messenger RNA In order for the new virus-specific proteins to be made from the virus genome, it is necessary for new virus-specific RNA molecules to be made. Exactly how the virus brings about new mRNA synthesis depends upon the type of virus, and especially upon whether its genetic material is RNA or DNA, and whether it is single-stranded or double-stranded. Which copy is read into mRNA depends upon the location of the appropriate promoter, since the promoter points the direction that the RNA polymerase will follow. In cells (uninfected with virus) all mRNA is made on the DNA template, but with RNA viruses the situation is obviously different. 

Reverse transcriptase is an RNA-dependent DNA polymerase that requires an RNA template to direct the synthesis of new DNA. Retroviruses, most notably HIV, use this enzyme to repHcate their RNA genomes. DNA synthesis by reverse transcriptase in retroviruses can be inhibited by AZT. ddC, and ddl. 

Furthermore, MSP analysis shonld not yield any prodncts nsing the same amount of unmodified genomic DNA as the template. The presence of products would indicate that the bisulfite treatment is incomplete, which more likely will generate a false-positive signal for methylation. This may also indicate that yonr primers are not specific for the modified DNA (see clues for designing primers for MSP, 9.3.4). 

The next stage is to ensure that the recombinant DNA molecule is copied by the enzymes which s)mthesize nucleic acids. These DNA and RNA polymerases synthesize an exact copy of either DNA or RNA from a pre-existing molecule. In this way the DNA polymerase duplicates the chromosome before each cell division such that each daughter cell will have a complete set of genetic instructions which are then passed to the newly formed RNA by RNA polymerase. While both DNA and RNA polymerase require a preformed DNA template, some viruses (such as HIV) have an RNA genome. To duplicate that genome, and incorporate it into a bacterial or mammalian cell, the viruses encode a reverse transcriptase enzyme which produces a DNA copy from an RNA template. 

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