分子生物学实验室技术

Vector DNAs typically have three characteristics：
1. They contain an origin of replication that allows them to replicate independently of the chromosome of the host. (Note that some yeast vectors also require a centromere.)
Figure 7-3
Figure 7-4
Recognition sequences and cut sites of various endonucleases
Figure 7-6
DNA Hybridization Can Be Used to Identify Specific DNA Molecules
• The living cell is an extraordinarily complicated entity, producing thousands of different macromolecules and harboring a genome that ranges in size from a million to billions of base pairs. • Understanding how the genetic processes of the cell work requires a variety of challenging experimental approaches.
• Annotation is the systematic identification of every stretch of genomic DNA that contains protein coding information or non-coding sequences that specify regulatory RNAs such as microRNAs.
Figure 7-3
Restriction Endonucleases Cleave DNA Molecules at Particular Sites
Restriction enzymes used in molecular biology typically recognize short (4–8 bp) target sequences, usually palindromic, and cut at a defined position within those sequences. EcoRI, so named because it was found in certain strains of Escherichia coli and was the first (I) such enzyme found in that species. This enzyme recognizes and cleaves the sequence 5′GAATTC-3′. (Because the two strands of DNA are complementary, we need specify only one strand and its polarity to describe a recognition sequence unambiguously.)
Vector DNA Can Be Introduced into Host Organisms by Transformation
• Transformation is the process by which a host organism can take up DNA from its environment. Some bacteria, but not E. coli, can do this naturally and are said to have genetic competence.
• These include methods for separating individual macromolecules from the myriad mixtures found in the cell and for dissecting the genome into manageably sized segments for manipulation and analysis of specific DNA sequences, as well as the use of suitable model organisms in which the tools of genetic analysis are available.
• The successful development of such methods has been one of the major driving forces in the field of molecular biology during the last several decades.
Figure 7-1
NUCLEIC ACIDS: BASIC METHODS
Gel Electrophoresis Separates DNA and RNA Molecules according to Size
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Figure 7-2
Pulsed-field gel electrophoresis
Chapter 3 Techniques of Molecular Biology
• Methods for the analysis of DNA and RNA; • The large-scale analysis of genomic DNA; • Analysis of proteins; • Large-scale analysis of proteins; • The analysis of nucleic acid–protein interactions.
Figure 7-8
• Some vectors not only allow the isolation and purification of a particular DNA but also drive the expression of genes within the insert DNA. • These plasmids are called expression vectors and have transcriptional promoters, derived from the host cell, immediately adjacent to the site of insertion.
• For the genomes of bacteria and simple eukaryotes, genome annotation is relatively straightforward, amounting essentially to the identification of open reading frames (ORFs). • For animal genomes with complex intron–exon structures, the challenge is far greater. In this case, a variety of bioinformatics tools are required to identify genes and determine the genetic composition of complex genomes.
2. They contain a selectable marker that allows cells that contain the vector (and any attached DNA) to be readily identified.
3. They have unique sites for one or more restriction enzymes. This allows DNA fragments to be inserted at a defined point within the vector such that the insertion does not interfere with the first two functions.
• Cells that have taken up the plasmid DNA— these cells are called transformants.
Figure 7-9
Libraries of DNA Molecules Can Be Created by Cloning
A DNA library is a population of identical vectors that each contains a different DNA insert . Genomic libraries and cDNA library.
Figure 7-10
Figure 7-11
Chemical Synthesis of Defined DNA Sequences
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DNA Sequencing
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Box 7-2
BOX 7-2 FIGURE 1 DNA sequence readout. In this reaction, as described in the text, fluorescently endlabeled dideoxynucleotides are used, and the chains are separated by column chromatography. The profile of positions of As is represented in green, Ts in red, Gs in black, and Cs in blue.
Figure 7-17
The Shotgun Strategy Permits a Partial Assembly of Large Genome Sequences
Figure 7-18
GENOMICS Bioinformatics Tools Facilitate the Genome-Wide Identification of Protein-Coding Genes
Figure 7-7
Isolation of Specific Segments of DNA DNA Cloning
•The ability to construct recombinant DNA molecules and maintain them in cells is called DNA cloning. •This process typically involves a vector that provides the information necessary to propagate the cloned DNA in the replicating host cell. Key to creating recombinant DNA molecules are the restriction enzymes that cut DNA at specific sequences and other enzymes that join the cut DNAs to one another. By creating recombinant DNA molecules that can be propagated in a host organism, a particular DNA fragment can be both purified from other DNAs and amplified to produce large quantities. •In a library, a common vector carries many alternative inserts. •Many common vectors are small (~3 kb) circular DNA molecules called plasmids.