CDNA fragmentation

Figure 2.19 Production of protein via a plasmid containing a cloned cDNA fragment. (Adapted with permission from Figure 3.8 of Lippard, Stephen J. Berg, Jeremy M. Principles of Bioinorganic Chemistry, University Science Books, Mill Valley, CA, 1994. Copyright 1994, University Science Books, Sausalito, CA.)...

King, D.P. et al., Molecular cloning and sequencing of interleukin 6 cDNA fragments from the harbor seal (Phoca vitulina), killer whale (Orcinus orca), and Southern sea otter (Enhydra lutris nereis), Immunogenetics, 43, 190, 1996. 

Generally differential display is used to compare levels of gene expression among experimental groups. However, this technique has also been used successfully to reveal mRNA sequence variation between samples. Eor example, differential display demonstrated that a cDNA fragment could be amphfied from samples derived from hippocampi of seizure-resistant but not seizure-prone rats (O Figure 17-2). A smaller... 

During their studies on OTA biosynthetic pathway genes, O Callaghan et al. (2003) did not only clone a polyketide synthase from A. ochraceus but cloned and sequenced several more differentially expressed cDNA fragments of yet unassigned origin from typical producers of OTA. Based on that work. University College Cork (Ireland) filed worldwide (WO 2004/072224 A2) as well as European (EP 1 592 705 A2)... 

A fully automated system uses either cDNA fragments of known genes or libraries of oligonucleotides, which are then spotted in duplicate onto hybridization filters (nylon membranes or PVDF) with a grid for guidance. When nylon membranes are used, these filters in microarrays can be used several times. There are two possible ways of applying the DNA to the filter (Cahill, 2001) ... 

Some viruses have secondary structure, which can prevent the production of cDNA detectable in a PCR assay by early termination of the synthesis reaction. To overcome this problem one can raise the temperature of incubation used in first-strand synthesis to 42°C or higher. This will reduce some secondary structures, but will also reduce the half-life of the reverse transcriptase. AMV reverse transcriptase may be used instead, because it has an optimal temperature of 42°C. Unfortunately, AMV RT has more endogenous RNaseH activity than M-MLV RT, thus on average AMV RT produces shorter cDNA fragments. RNaseH deficient RT enzymes are also available (e.g., the Superscript enzymes from Invitrogen), and there is some evidence that these may be the most sensitive type of RT enzymes for PCR assays. The RT conditions required for the efficient detection of individual viruses can only be determined empirically. 

Figure 2 Assembly of human delta opioid receptor open reading frame. The figure illustrates how the human delta opioid receptor open reading frame was assembled from the 44-11 and 78-4 cDNA fragments using the Hindi and Apal restriction sites.

The protein pVI is one of the minor coat proteins present at five copies per phage in one tip of the virion [20]. Its function is to connect the phage body to pill protein and expose the C terminus to the solvent. Jespers and co-workers [21] have constructed a new vector in which they inserted cDNA fragments fused to the gene 6. 

Fig. 4.24. Hypothetical scheme for cloning cDNA fragments from reverse transcripts of RNA into M13 vectors for sequencing.

For purification of cDNA fragments from agarose gels PCR Prep Kit (Promega). 

Recover the cDNA fragments ranging in size from 300-600 bp by PCR Prep Kit. 

Ligation of the cDNA Fragments to the pDREF-CD4ST Vector... 

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