From Genes to Genomes:- DNA Libraries Provide Specialized Catalogs of Genetic Information

A DNA library is a collection of DNA clones, gathered together as a source of DNA for sequencing, gene discovery, or gene function studies. The library can take a variety of forms, depending on the source of the DNA. Among the largest types of DNA library is a genomic library, produced when the complete genome of a particular organism is cleaved into thousands of fragments, and all the fragments are cloned by insertion into a cloning vector.

The first step in preparing a genomic library is partial digestion of the DNA by restriction endonucleases, such that any given sequence will appear in fragments of a range of sizes—a range that is compatible with the cloning vector and ensures that virtually all sequences are represented among the clones in the library. Fragments that are too large or too small for cloning are re moved by centrifugation or electrophoresis. The cloning vector, such as a BAC or YAC plasmid, is cleaved with the same restriction endonuclease and ligated to the ge nomic DNA fragments. The ligated DNA mixture is then used to transform bacterial or yeast cells to produce a library of cell types, each type harboring a different recombinant DNA molecule. Ideally, all the DNA in the genome under study will be represented in the library. Each transformed bacterium or yeast cell grows into a colony, or “clone,” of identical cells, each cell bearing the same recombinant plasmid.

Using hybridization methods, researchers can order individual clones in a library by identifying clones with overlapping sequences. A set of overlapping clones rep resents a catalog for a long contiguous segment of a genome, often referred to as a contig (Fig. 9–13). Previously studied sequences or entire genes can be located within the library using hybridization methods to determine which library clones harbor the known sequence. If the sequence has already been mapped on a chromosome, investigators can determine the location (in the genome) of the cloned DNA and any contig of which it is a part. A well-characterized library may contain thousands of long contigs, all assigned to and or dered on particular chromosomes to form a detailed physical map. The known sequences within the library (each called a sequence-tagged site, or STS) can provide landmarks for genomic sequencing projects. As more and more genome sequences become available, the utility of genomic libraries is diminishing and investigators are constructing more specialized libraries designed to study gene function. An example is a library that includes only those genes that are expressed—that is, are transcribed into RNA—in a given organism or even in certain cells or tissues. Such a library lacks the noncoding DNA that makes up a large portion of many eukaryotic genomes. The researcher first extracts mRNA from an organism or from specific cells of an or ganism and then prepares complementary DNAs (cDNAs) from the RNA in a multistep reaction cat alyzed by the enzyme reverse transcriptase (Fig. 9–14). The resulting double-stranded DNA fragments are then inserted into a suitable vector and cloned, creating a population of clones called a cDNA library. The search for a particular gene is made easier by focusing on a cDNA library generated from the mRNAs of a cell known to express that gene. For example, if we wished to clone globin genes, we could first generate a cDNA library from erythrocyte precursor cells, in which about half the mRNAs code for globins. To aid in the mapping of large genomes, cDNAs in a library can be partially sequenced at random to produce a useful type of STS called an ex pressed sequence tag (EST). ESTs, ranging in size from a few dozen to several hundred base pairs, can be positioned within the larger genome map, providing markers for expressed genes. Hundreds of thousands of ESTs were included in the detailed physical maps used as a guide to sequencing the human genome.

FIGURE 9–13 Ordering of the clones in a DNA library. Shown here is a segment of a chromosome from a hypothetical organism X, with markers A through Q representing sequence-tagged sites (STSs—DNA segments of known sequence, including known genes). Below the chromosome is an array of ordered BAC clones, numbered 1 to 9. Ordering the clones on the genetic map is a many-stage process. The presence or absence of an STS on an individual clone can be determined by hybridization—for example, by probing each clone with PCR-amplified DNA from the STS. Once the STSs on each BAC clone are identified, the clones (and the STSs themselves, if their location is not yet known) can be ordered on the map. For example, compare clones 3, 4, and 5. Marker E (blue) is found on all three clones; F (red) on clones 4 and 5, but not on 3; and G (green) only on clone 5. This indicates that the order of the sites is E, F, G. The clones partially over lap and their order must be 3, 4, 5. The resulting ordered series of clones is called a contig.

A cDNA library can be made even more specialized by cloning a cDNA or cDNA fragment into a vector that fuses the cDNA sequence with the sequence for a marker, or reporter gene; the fused genes form a “re porter construct.” Two useful markers are the genes for green fluorescent protein and epitope tags. A target gene fused with a gene for green fluorescent protein (GFP)generates a fusion protein that is highly fluorescent—it literally lights up (Fig. 9–15a). Just a few molecules of this protein can be observed microscopically, allowing the study of its location and movements in a cell. An epitope tagis a short protein sequence that is bound tightly by a well-characterized monoclonal antibody (Chapter 5). The tagged protein can be specifically precipitated from a crude protein extract by in teraction with the antibody (Fig. 9–15b). If any other proteins bind to the tagged protein, those will precipitate as well, providing information about protein-protein interactions in a cell. The diversity and utility of specialized DNA libraries are growing every year.

FIGURE 9–14 Construction of a cDNA library from mRNA.A cell’s mRNA includes transcripts from thousands of genes, and the cDNAs generated are correspondingly heterogeneous. The duplex DNA produced by this method is inserted into an appropriate cloning vector. Reverse transcriptase can synthesize DNA on an RNA or a DNA template (see Fig. 26–29).

اخر الاخبار

اخبار العتبة العباسية المقدسة

قسم الشؤون الخدمية ينفذ حملة لتنظيف الشوارع المحيطة بصحن مرقد أبي الفضل العباس (عليه السلام)

وفد العتبة العباسية المقدسة يزور شعبة الإعداد البدني للاطّلاع على برنامج تأهيل المنتسبين الجدد

قسم بين الحرمين يقدم جهودًا مضاعفة لإعادة المفقودات إلى الزائرين

بعد برامج تدريبية متخصصة.. متحف الكفيل يبيّن منجزات ملاكاته في فن الخاتم (الأرابيسك)

المزيد

الآخبار الصحية

ماذا يحدث إذا تناولنا الطعام مرة واحدة فقط في اليوم

اكتشاف عوامل خطر غير متوقعة لمرض الكبد الدهني

فيروس هانتا والسفينة الموبوءة.. تحديث من الصحة العالمية

طبيب يحسم الجدل السجائر الإلكترونية وعلاقتها بالسرطان

المزيد

الآخبار التكنلوجية

لأول مرة في الولايات المتحدة.. تشغيل مفاعل مصغر نووي يغذي الذكاء الاصطناعي بالطاقة

الكشف عن تفاصيل أول عملية ناجحة لاستعادة انتاج الحيوانات المنوية

أطلس يكشف خفايا الجسم البشري بدقة أرفع بـ50 مرة من شعر الإنسان

المتحف المصري الكبير يتحول إلى "أيقونة خضراء" بدعم ياباني

المزيد

مواضيع ذات صلة

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From Genomes to Proteomes:- Detection of Protein-Protein Interactions Helps to Define Cellular and Molecular Function

From Genomes to Proteomes:- Cellular Expression Patterns Can Reveal the Cellular Function of a Gene

From Genomes to Proteomes:- Sequence or Structural Relationships Provide Information on Protein Function

From Genes to Genomes:- Genome Sequences Provide the Ultimate Genetic Libraries

From Genes to Genomes:- The Polymerase Chain Reaction Amplifies Specific DNA Sequences

DNA Cloning: The Basics:- Alterations in Cloned Genes Produce Modified Proteins