遗传学实验技术

The extraordinary sensitivity of reverse-transcriptase-polymerase chain reaction (RT-PCR) makes it a powerful technique for specific mRNA detection, particularly when tissue availability is limiting, and when the mRNA to be detected is present in low abundance. A disadvantage ...

The completion of whole-genome sequencing projects offers the opportunity of creating high-resolution maps of specific segments in a known genomic DNA sequence. For this purpose, several genome browsers have been created. They include the map-view (http:// www.ncbi.nlm.nih.gov/m ...

At any particular point in time, the full complement of transcribed RNAs and relevant proteins of a cell are known as the transcriptome and proteome, respectively. The composition of these two populations changes throughout the life cycle of a parasite or in response to environmental factors, ...

DNA microarray platforms represent a functional genomics technology that uses structured information obtained from genomic sequencing efforts as a means to study transcriptional processes in a systematic and high-throughput manner. Specifically in this chapter, we outline ...

The genome sequencing of protozoan parasites has facilitated the development of powerful postgenomics tools such as DNA microarrays and revolutionized the study of parasite biology. Large-scale genomic comparisons are useful in identifying the extent of genomic variability a ...

Genotyping is an important tool for epidemiological and population genetic studies in protozoan parasites. The most commonly used method for genotyping is polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis of single nucleotide p ...

Most members of the AID/APOBEC family of polynucleotide deaminases can catalyse the deamination of cytosine to uracil in DNA. They thereby function as active DNA mutators. Here, we describe how bacterial papillation assays can be adapted to monitor the mutator activity of AID/APOBEC prot ...

Human APOBEC3G (A3G) is a cytidine deaminase that broadly restricts the replication of many retroviruses, including HIV-1. In different cell types, cytoplasmic A3G is expressed in high-molecular-mass (HMM) RNA–protein complexes or low-molecular-mass (LMM) forms displaying dif ...

RNA editing deaminases act on a variety of targets in different organisms. A number of such enzymes have been shown to act on mRNA, with the resultant nucleotide changes modifying a transcript’s information content. Though the deaminase activity of mRNA editing enzymes is readily demonstra ...

Substitutional RNA editing represents an important posttranscriptional enzymatic pathway for increasing genetic plasticity by permitting production of different translation products from a single genomically encoded template. One of the best-characterized examp ...

mRNA editing in plastids (chloroplasts) of higher plants proceeds by cytidine-to-uridine conversion at highly specific sites. Editing sites are recognized by the interplay of cis-acting elements at the RNA level and site-specific trans-acting protein factors that are believed to b ...

The multiplex single base extension SNP-typing procedure outlined here can be employed to screen large numbers of plants for mutations in nuclear genes that affect mitochondrial RNA editing. The high �sensitivity of this method allows high-throughput analysis of individual plants ...

The primary sequence of all nucleic acids in a cell contain 4 canonical nucleotides (G, A, T, and C for DNA and G, A, U, and C for RNA). However, post-transcriptionally, nucleic acids can undergo a number of chemical modifications, which may change their structure and function. tRNAs contain the most diverse ...

The recent discovery of thousands of small noncoding RNAs (ncRNAs), in many different organisms, has led to the need for methods to study their function. One way to help understand their function is to determine what other RNAs interact with the ncRNAs. We have developed a novel method to investigate ...

RNA-guided RNA 2′-O-methylation and pseudouridylation are naturally occurring processes, in which guide RNAs specifically direct modifications to rRNAs or spliceosomal snRNAs in the nucleus of eukaryotic cells. Modifications can profoundly alter the properties of an RNA, thus ...

Deoxyribozymes or DNAzymes are small DNA molecules with catalytic activity originating from in vitro selection experiments. Variants of the two most popular DNAzymes with RNase activity, the 10–23 DNAzyme and the 8–17 DNAzyme, promote efficient in vitro cleavage of the phosphodiest ...

Here we describe a LightCycler-based real-time PCR for quantitative detection of EBV DNA in clinical samples such as unfractionated whole blood, serum, or plasma. This assay is based on amplification of a highly conserved 213-bp region of the EBNA-1 gene, a single-copy gene of EBV required for mai ...

The focus of this chapter is the detection of DNA viruses. The emphasis is on amplification reactions that include reverse transcription-polymerase chain reaction (RT-PCR), PCR, real-time RT-PCR, and real-time PCR methods. Amplification of the E6 and E7 oncoproteins of HPV16 is described ...

We describe a two-step RT-PCR method for simultaneous detection of EBNA-1 (QK and Y3K splice variants), EBNA-2, LMP-1, LMP-2a and -2b, ZEBRA, and BARTs RNA encoded by Epstein-Barr virus. As a control for RNA integrity, the low-copy-number transcript derived from U1 A snRNP, a cellular housekeeping ge ...

The method described in this chapter uses limiting dilution analysis in conjunction with RT-PCR to determine quantitatively what percentage of EBV-infected cells within a given population are expressing the viral genes EBNA-1 Q-K, EBNA-2, LMP-1, LMP-2, BZLF-1, and the EBERs. Because this te ...