遗传学实验技术

Precise and accurate determination of mRNA expression levels in tissues and model systems is a central methodology in a wide range of research applications. Expression of many genes is currently assessed by northern blotting, RNAse protection assays, Serial Analysis of Gene Expressi ...

The use of quantitative polymerase chain reaction (qPCR) to detect RNA viruses has become increasingly important as a prognostic marker and in patient management, for example, in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. Drug therapies can be monitored by r ...

Chromosomal aberrations, such as translocations or inversions, described for a growing number of malignancies, are now widely used to detect tumor cells by polymerase chain reaction (PCR). However, in multiple myeloma (MM), no such ubiquitous PCR marker exists. Therefore, other means ha ...

The nature of the polymerase chain reaction (PCR) process lends itself well to qualitative determinations. It transforms very small quantities of analyte into the realms of bucket chemistry, allowing specific gene portions to be directly visualized with ethidium bromide and ultrav ...

In this chapter, we detail protocols of long polymerase chain reaction (PCR) and long RT-PCR, which we have found to be versatile, sensitive, and straightforward to optimize. We have used these protocols with success on several different templates, including lambda phage DNA, HAV, HBV, HCV (1), to ...

The polymerase chain reaction (PCR) has become an essential and ubiquitous tool for biological research and laboratory diagnostic applications. Until recently, reliable and sensitive amplification of large templates (several kb) was difficult to achieve. However, in 1994, an imp ...

Nucleic acids used for polymerase chain reaction (PCR) assays usually are extracted by the phenol-chloroform method or an alternative rapid purification. The acid-guanidinium thiocyanate-phenol-chloroform method for RNA extraction and proteinase K digestion-phenol-c ...

Single nucleotide polymorphisms (SNPs) are single base differences in genomic DNA (1). These single-base mutations, estimated to occur every 1000 bases, are thought to represent the most common form of genetic variation in the human genome (2). Several million SNPs have been identified (3). H ...

The study of genetic polymorphism among diverse populations of organisms is a complex task. However, this can be accomplished by using newer tools, such as randomly amplified polymorphic DNA (RAPD). RAPD is a polymerase chain reaction (PCR) technique that relies on the generation of amplifi ...

Hemophilia A is an X-linked disorder caused by mutations in the factor VIII gene. Around 50% of all patients with severe hemophilia A share a common mutation. This intron 22 inversion results from homologous recombination of a sequence within intron 22 of the factor VIII gene and identical sequence ...

There are several methods now in widespread use for detecting and characterizing specific RNA targets. These methods include in situ hybridization, Northern blotting, dot or slot blot, RNase protection assay, and reverse transcription coupled to polymerase chain reaction (RT-PCR). ...

Analysis of DNA variation (polymorphism and mutations) is one of the most common challenges faced by molecular biologists. Studies of polymorphisms and mutations as molecular markers of or underlying causes of disease have confirmed the importance of mutation and polymorphism dete ...

Many gene-cloning strategies and gene survey often provide partial sequence data. To exploit the information from these partial sequences numerous PCR-based approaches have been developed to clone full-length open reading frames. These approaches can be successful using small q ...

Parasites successfully exist within the host as a result of highly specific genetic adaptations. Therefore, detecting genes that contain relevant adaptive mutations can provide a guide to biological processes that are potentially essential to the parasite. Random genetic mutat ...

Expressed sequence tags (ESTs) present a special set of problems for bioinformatic analysis. They are partial and error-prone, and large datasets can have significant internal redundancy. To facilitate analysis of small EST datasets from in-house projects, we present an integrated “ ...

Generating expressed sequence tags is a simple, cheap, and efficient way to sample the genome of a target organism. An expressed sequence tag (EST) is a single-pass sequence derived from a single complementary DNA (cDNA) clone, and the sequence serves to identify the gene from which it derives. We pre ...

In the last decade, high-throughput genome sequencing and complementary techniques such as microarray and proteomics have generated, and will continue to generate, ever-increasing amounts of data. These technologies of gene discovery, expression, and functional analysis have ...

Genome annotation is the application of useful biological descriptions to sequence data. Different levels of time and effort can be invested to produce correspondingly different depths of annotation depending on what methods are employed. Researchers using genome data should, th ...

A total of 2183 clones derived from four life cycle stage-specific, spliced-leader cDNA libraries of Leishmania major LV39 Neal strain were randomly picked and sequenced to generate expressed sequence tags (ESTs). Then 1094 unique genes were identified, with 18.2% having BLAST hits with kn ...

Protein-protein interactions occur in a wide variety of biological processes and essentially control the cell fate from division to death. Today, the identification of proteins that interact with a protein of interest is a focus of intensive research and is an essential element of the rapid ...