Harrison's Internal Medicine. A description of recombinant DNA techniques, the methodology used for the manipulation, analysis, and characterization of DNA segments, is beyond the scope of this chapter. As these methods are widely used in genetics and molecular diagnostics, however, it is useful to review briefly some of the fundamental principles of cloning and DNA sequencing. Cloning of Genes.

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Harrison's Internal Medicine. A description of recombinant DNA techniques, the methodology used for the manipulation, analysis, and characterization of DNA segments, is beyond the scope of this chapter. As these methods are widely used in genetics and molecular diagnostics, however, it is useful to review briefly some of the fundamental principles of cloning and DNA sequencing. Cloning of Genes. Cloning refers to the creation of a recombinant DNA molecule that can be propagated indefinitely.

The ability to clone genes and cDNAs therefore provides a permanent and renewable source of these reagents. Cloning is essential for DNA sequencing, nucleic acid hybridization studies, expression of recombinant proteins, and other recombinant DNA procedures. The most straightforward cloning strategy involves inserting a DNA fragment into bacterial plasmids. Plasmids are small, autonomously replicating, circular DNA molecules that propagate separately from the chromosome in bacterial cells.

The process of DNA insertion relies heavily on the use of restriction enzymes, which cleave DNA at highly specific sequences usually 4—6 bp in length. Restriction enzymes generate complementary, cohesive sequences at the ends of the DNA fragment, which allow them to be efficiently ligated to the plasmid. Because plasmids contain genes that confer resistance to antibiotics, their presence in the host cell can be used for selection and DNA amplification.

A variety of vectors e. Many of these are used for creating libraries , a term that refers to a collection of DNA clones. A genomic library represents an array of clones derived from genomic DNA. These overlapping DNA fragments represent the entire genome and can ultimately be arranged according to their linear order.

Thus, a cDNA library from the heart contains copies of mRNA expressed specifically in cardiac myocytes, in addition to those that are expressed ubiquitously.

As an example of the complexity of a genomic library, consider that the human genome contains 3 x. Therefore, it requires at least 3 x 10 5 clones to represent all genomic DNA. Specific clones are isolated from the several hundred thousand clones by using DNA hybridization. With completion of the HGP, all human genes have been cloned and sequenced. As a result, many of these cloning procedures are now unnecessary or greatly facilitated by the extensive information concerning DNA markers and the sequence of DNA see below.

Nucleic acid hybridization is a fundamental principle in molecular biology that takes advantage of the fact that the two complementary strands of nucleic acids bind, or hybridize , to one another with very high specificity. The goal of hybridization is to detect specific nucleic acid DNA or RNA sequences in a complex background of other sequences. This technique is used for Southern blotting, Northern blotting, and for screening libraries see above.

Southern blotting is used to analyze whether genes have been deleted or rearranged. It is also used to detect restriction fragment length polymorphisms RFLPs. Genomic DNA is digested with restriction endonucleases and separated by gel electrophoresis. Individual fragments can then be transferred to a membrane and detected after hybridization with specific radioactive DNA probes.

Because single base-pair mismatches can disrupt the hybridization of short DNA probes oligonucleotides , a variation of the Southern blot, termed.

Northern blots are used to analyze patterns and levels of gene expression in different tissues. In a Northern blot, mRNA is separated on a gel and transferred to a membrane, and specific transcripts are detected using radiolabeled DNA as. This technique has been largely supplanted by more sensitive and comprehensive methods such as reverse transcriptase RT —PCR and gene expression arrays on DNA chips see below.

A comprehensive approach to genome-scale studies consists of microarrays , or. DNA chips. These microarrays consist of thousands of synthetic nucleic acid sequences aligned on thin glass or silicon surfaces. Microarrays allow the detection of variations in DNA sequence and are used for mutational analysis and genotyping. This method has tremendous potential in the era of functional genomics and. As one example, microarrays can be used to develop genetic fingerprints of different types of malignancies, providing information useful for classification, pathophysiology, prognosis, and treatment.

The PCR, introduced in , has revolutionized the way DNA analyses are performed and has become a cornerstone of molecular biology and genetic analysis. The geometric amplification of the DNA after multiple cycles yields remarkable sensitivity.

These properties also allow DNA amplification from a variety of tissue sources including blood samples, biopsies, surgical or autopsy specimens, or cells from hair or saliva. PCR provides a key component of molecular diagnostics.

PCR is also used for the amplification of highly polymorphic di- or trinucleotide repeat sequences or the genotyping of SNPs, which allow various polymorphic alleles to be traced in genetic linkage or association studies. PCR is increasingly used to diagnose various microbial pathogens. DNA sequencing is now an automated procedure. Although many protocols exist, the most commonly used strategy currently uses the capillary electrophoresis-based Sanger method in which dideoxynucleotides are used to randomly terminate DNA polymerization at each of the four bases A,G,T,C.

After separating the array of terminated DNA fragments using high-resolution gel or capillary electrophoresis, it is possible to deduce the DNA sequence by examining the progression of fragment lengths generated in each of the four nucleotide reactions.

The use of fluorescently labeled dideoxynucleotides allows automated detection of the different bases and direct computer analysis of the DNA sequence Fig. Significant efforts are underway to develop faster, more cost-effective DNA sequencing technologies. These include the use of pyrosequencing chemistries; whole-genome sequencing using solid-phase sequencing; mass spectrometry; detection of fluorescently labeled bases in flow cytometry; direct reading of the DNA sequence by scanning, tunneling, or atomic force microscopy; and sequence analysis using DNA chips.

Transgenic Mice as Models of Genetic Disease. Several organisms have been studied extensively as genetic models, including. Mus musculus mouse , Drosophila melanogaster fruit fly , Caenorhabditis elegans nematode , Saccharomyces cerevisiae baker's yeast , and.

Escherichia coli colonic bacterium. The ability to use these evolutionarily distant organisms as genetic models that are relevant to human physiology reflects a surprising conservation of genetic pathways and gene function. Transgenic mouse models have been particularly valuable, because many human and mouse genes exhibit similar structure and function, and because manipulation of the mouse genome is relatively straightforward compared to those of other mammalian species.

Transgenic strategies in mice can be divided into two main approaches: 1 expression of a gene by random insertion into the genome, and 2 deletion or targeted mutagenesis of a gene by homologous recombination with the native endogenous gene knock-out, knock-in Fig.

Transgenic mice are generated by pronuclear injection of foreign DNA into fertilized mouse oocytes and subsequent transfer into the oviduct of pseudopregnant foster mothers. The HGP was initiated in the mids as an ambitious effort to characterize the human genome, culminating in a complete DNA sequence. The initial main goals were 1 creation of genetic maps, 2 development of physical maps, and 3 determination of the complete human DNA sequence. Some analogies help in appreciating the scope of the HGP.

If the human DNA sequence were printed out, it would correspond to about volumes of Harrison's Principles of. Internal Medicine. SNPs that are in close proximity are inherited together, i. The HapMap describes the nature and location of these SNP haplotypes and how they are distributed among individuals within and among populations. The HapMap information is greatly facilitating genome-wide association studies designed to elucidate the complex interactions among multiple genes and lifestyle factors in multifactorial disorders see below.

Moreover, haplotype analyses may become useful to assess variations in responses to medications pharmacogenomics and environmental factors, as well as the prediction of disease predisposition. The complete DNA sequence of each chromosome provides the highest resolution physical map. Although the prospect of determining the complete sequence of the human genome seemed daunting several years ago, technical advances in DNA sequencing and bioinformatics led to the completion of a draft human sequence in June , well in advance of the original goal year of High-quality reference sequences, completed in ,.

They include, among others, eukaryotes such as. This information, together with technological advances and refinement of computational bioinformatics, has led to a fast-paced transition from the study of single genes to whole genomes.

The current directions arising from the HGP include, among others, 1 the comparison of entire genomes comparative genomics , 2 the study of large-scale expression of RNAs functional genomics and proteins proteomics in order to detect differences between various tissues in health and disease, 3 the characterization of the variation among individuals by establishing catalogues of sequence variations and SNPs HapMap project , and 4 the identification of genes that play critical roles in the development of polygenic and multifactorial disorders.

Implicit in the HGP is the idea and hope that identifying disease-causing genes can lead to improvements in diagnosis, treatment, and prevention. It is estimated that most individuals harbor several serious recessive genes. However, completion of the human genome sequence, determination of the association of genetic defects with disease, and studies of genetic variation raise many new issues with implications for the individual and mankind. The controversies concerning the cloning of mammals and the establishment of human ES cells underscore the relevance of these questions.

Moreover, the information gleaned from genotypic results can have quite different impacts, depending on the availability of strategies to modify the course of disease. For example, the identification of mutations that cause multiple endocrine neoplasia MEN type 2 or hemochromatosis allows specific interventions for affected family members. On the other hand, at present, the identification of an Alzheimer or Huntington disease gene does not alter therapy.

In addition, the progress in this area is unpredictable, as underscored by the finding that angiotensin II receptor blockers may slow disease progression in Marfan syndrome. Genetic test results can generate anxiety in affected individuals and family members, and there is the possibility of discrimination on the basis of the test results.

Most genetic disorders are likely to fall into an intermediate category where the opportunity for prevention or treatment is significant but limited Chap. For these reasons, the scientific components of the HGP have been paralleled by efforts to examine ethical and legal implications as new issues arise.

Many issues raised by the genome project are familiar, in principle, to medical practitioners. For example, an asymptomatic patient with increased low-density lipoprotein LDL cholesterol, high blood pressure, or a strong family history of early myocardial infarction is known to be at increased risk of coronary heart disease. In such cases, it is clear that the identification of risk factors and an appropriate intervention are beneficial.

Likewise, patients with phenylketonuria, cystic fibrosis, or sickle cell anemia are often identified as having a genetic disease early in life. These precedents can be helpful for adapting policies that relate to genetic information.

We can anticipate similar efforts, whether based on genotypes or other markers of genetic predisposition, to be applied to many disorders. One confounding aspect of the rapid expansion of information is that our ability to make clinical decisions often lags behind initial insights into genetic mechanisms of disease. For example, when genes that predispose to breast cancer, such as BRCA1 , are described, they generate tremendous public interest in the potential to predict disease, but many years of clinical research are still required to rigorously establish genotype and phenotype correlations.

Whether related to informed consent, participation in research, or the management of a genetic disorder that affects an individual or their families, there is a great need for more information about fundamental principles of genetics. The pervasive nature. The application of screening and prevention strategies will therefore require intensive patient and physician education, changes in health care financing, and legislation to protect patient's rights.

For the practicing clinician, the family history remains an essential step in recognizing the possibility of a hereditary component. When taking the history, it is useful to draw a detailed pedigree of the first-degree relatives e. Standard symbols for pedigrees are depicted in Fig.


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