Techniques of Molecular Biology

Chapter 20

生科 1 班 王婧 200332540269

OUTLINE

Introduction(p.647)Nucleic Acids(p.648)Proteins(p.672)

INTRODUCTION

Understanding how the genetic processes of the cell work requires powerful,and complementary experimental approaches including the use of suitable model organisms in which the tools of genetic analysis are available.

Methods for separating individual macromolecules from the myriad mixtures found in the cell,and for dissecting the genome specific DNA sequences

Using computational or computational or bioinformatics approaches,to undertake large-scale genomic comparisons of both the cosing and noncoding regions of various organisms.The methods of molecular biology depend upon,and were developed from,an understanding of the properties of biological macromolecules themselves.

A NOTE:It is important to appreciate that when

we talk about isolating and purifying a given macromolecule in the ensuing discussion we rarely mean that a single molecule is isolated.rather,the goal of these procedures is to isolate a large population of identical molecules from all of the other kinds of molecules in cell

NUCLEIC ACIDS

This part is devoted to techniques for the manipulation and characterization of nucleic acids,from the isolation of RNAs and DNAs to the sequencing of entire genomes and comparative genomics.

1.1 Electrophoresis through a Gel separates DNA and RNA Molecules According to Size

Gel electrophoresis ( 凝胶电泳 ) separation of DNA and RNA molecules.Linear DNA molecules separate according to size when subject to an electric field through a gel matrix(an inert,jello-like porous material) 动画

DNA molecules are flexible and occupy an effective volume pores in the gel matrix sieve the DNA molecules according to this volume;large molecules migrate more slowly through the gel because they have a larger effective volume than do smaller DNAs,and thus have more difficulty passing through the interstices of the gel.

Different sizes are separated because they moved different distances through the gel.

Figure 20-1 DNA separation by gel electrophoresis

After electrophoresis is complete:DNA molecules can be visualized by staining th

e gel with fluorescent dyes(such as ethidium溴化乙锭 )

Two alternative kinds of gel matrices: Polyacrylamide( 聚丙烯酰胺 ):has high resolvin

g capability but can separate DNAs only over a narrow size range. Can resolve DNAs that differ from each other in size by as little as a single base pair but only with molecules of up to several hundred base pairs.

Agarose( 琼脂糖 )has less resolving power than polyacrylamide but can separate from one another DNA molecules of up to tens,and even hundreds,of kilobases.

Pulsed-field gel electrophoresis( 脉冲电场凝胶电泳 )very long DNAs be resolved from one another if the electric field is applied in pulses that are oriented orthogonally to each other.figure20-2

Each time the orientation of the electric field changes,the DNA molecule,snaking its way through the gel,reorient to the direction of the new field.the larger the DNA,the longer it takes to reorient.

This can be used to determine the size of entire bacterial chromosomes and chromosomes of lower eukaryotes.

Figure 20-2 pulsed-field gel electrophoresis

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Electrophoresis separates DNA molecules according to their molecular weight,shape topological properties. (figure 6-26)

It is used to separate RNAs.RNAs have a uniform negative charge,but RNA usually single-stranded and have extensive secondary and tertiary structure.to deal with this ,RNAs can be treated with reagents to prenvent the formation of base pairs.

Figure 6-26 schematic of electrophoretic separation of DNA topoisomers.

1.2 Restriction Endonucleases Cleave DNA Molecules at

Particular SitesRestriction endonucleases:nucleases that

cleave DNA at particular sites by the recognition of specific sequences.

Restriction enzymes used in molecular biology typically recognize short(4-8bp) target sequences,usually palindromic,and cut at a defined position within those suquences.

Example:EcoR1 recognizes and cleaves the sequence

5’-GAATTC-3’(figure 20-3)Sau3A1 recognizes an octameric sequence5’-

GCGGCCGC-3’ and cut.(table 20-1)

Figure 20-3 Digestion of a DNA fragment with endonuclease EcoRI

Table 20-1 Some Restriction Endonucleases and Their Recognition Sequences

Enzyme Sequence Cut frequency

Sau3A1 5`-GATC-3` 0.25kb

EcoR1 5`-GATTC-3` 4kb

Not1 5`-GCGGCCGC-3` 65kb

Figure 20-4 Recognition sequences and cut sites of various endonucleases

Different endonucleases recognize different traget sites,cut at different positions within those sites. Molecules with blunt ends or with 5`or 3` overhanging ends can be generated.

Figure 20-5 Cleavage of an EcoRI site

1.3 DNA Hybridization Can Be Used to Identify Specific DNA

MoleculesHybridization( 杂交 ): The capacity of denatured to reanneal allows f

or the formation of hybrid molecules homologous, denatured DNAs from two different sources are mixed with each other under the appropriate conditions of ionic strength and temperature. the process of base-pairing between complementary single-stranded polynucleotides from two different sources.

Probe( 探针 )be used to search mixtures of nucleic acids for molecules containing a complementary sequence. The pro DNA must be labeled so that it can be readily located once it had found its target sequence.

There are two basic methods for labeling DNA.the first involves synthesizing new DNA in the presence of a labeled precursor;the other involved adding a label to the end of an intact DNA molecule.

1.4 Hybridization Probes Can Identify Electrophoretically Separated DNAs and RNAs

Southern blot hybridization( 印迹杂交 )identify within the smear the size of the particular fragment containing your gene of interest.

The cut DNA that has been separated by gel electrophoresis is soaked in alkali to denature the double-stranded DNA fragments.transferred to a positively-charged membrane.

The DNA bound to the membrane incubated with probe DNA containing a sequence complementary to a sequence within the gene of interest.

Autoradiogram( 放射自显影 ):on the blot the probe hybridizes detected by a variety of films or other media that are sensitive to the light or electrons emitted by the labeled DNA .

Figure 20-6 A Southern Blot

Northern blot hybridization be used to identify a particular mRNA in a population of RNAs.

Use northern blot hybridization to ask how much more mRNA of a specific type is present in a cell treated with an inducer of the gene in question compared to an uninduced cell.

1.5 Isolation of Specific Segments of DNA

Much of the molecular analysis of genes and their function requires the separation of specific segments of DNA from much larger DNA molecules, and their selective.

DNA cloning and amplification by PCR have become essential tools in asking questions about the control of gene expression and maintenance of the genome.

1.6 DNA Cloning

DNA cloning:to construct recombinant DNA molecules and maintain them in cells.

This process typically involves a vector that provides the information necessary to propagate the cloned DNA in the cell and an insert DNA that is inserted within the vector and includes the DNA of interest

1.7 Cloning DNA in Plasmid VectorsThe DNA fragment must be inserted within that secon

d DNA molecule to be replicated in a host organism.Vector DNAs typically have three characteristics: They contain an origin of replication that allows the

m to replicate independently of the chromosome of the host.

They contain a selectable marker that allows cells that contain the vector to be readily identified.

They have single sites for one or more restriction enzymes.this allows DNA fragments to be inserted at a defined point within an otherwise intact vector.

Plasmids( 质粒 ):the most common vectors are small circular DNA molecules. Derived from circular DNA molecules. Found naturally in many bacteria and single-cell eukaryotes.

Two characteristics:Can propagate independently in the host,car

ry a selectable marker.Present in multiple copies per cell. This incre

ases the amount of DNA that can be isolated from a population.

To insert a fragment of DNA into a vector is a relatively simple process(figure 20-7):

Restriction enzyme linearize the plasmid.A restriction enzyme cleaved a target DNA to

generate potential insert DNAs,vector DNA has been cut with the same enzyme.

DNA ligase to link the compatible ends of the two DNAs.

Figure 20-7 Cloning in a plasmid vector

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:Used to express heterologous or mutant gen

es to assess their function.Used to produce large amounts of a protein f

or purification

1.8 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.

The bacteric have genetic competence means do transformation naturally.

Transformation generally is a relatively inefficientprocess.

It is this low efficiency of transformation that:Makes necessary selection with the antibiotic.Also ensures ,in most cases,each cell receives

only a single molecule of DNA.

1.9 Libraries of DNA Molecules Can Be Created by Cloning

A DNA library( 文库 )is a population of identical vectors that each contains a different DNA insert.Figure 20-8

Genomic libraries( 基因组文库 ) derived from total genomic DNA cleaved with a restriction enzyme.

Reverse transcription( 反转录 ) to enrich for coding sequences in the library,a cDNA library is used.Figure 20-9

mRNA is converted into DNA sequence.performed by a special DNA polymerase that can make DNA from an RNA template.mRNA sequences can be converted into double-stranded DNA-----cDNAs.

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Figure 20-8 Construction of a DNA library

Figure 20-9 Constuction of a cDNA library

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1.10 Hybridization Can Be Used to Identify a Specific Clone in a DNA

Library

Using a DNA probe whose sequence matches part of the gene of interest.such a probe can be used to identify colonies of cells harboring clones containing that region of the gene.

Colony hybridization a labeled DNA probe id used to screen a library.

1.11 Chemically Synthesized Oligonucleotides

Phosphoamidines: the most common methods of chemical synthesis are performed on solid supports using machines that automate the process. The precursors used for nucleotide addition are chemically protected molecules.Figure 20-10

Site-directed mutagenesis the oligonucleotide is hybridized to the cloned fragment,and used to prime DNA synthesis with the cloned DNA as template. A double-stranded molecule with one mismatch id made. The two strands are then separated and that with the desired mismatch amplified further.

Figure 20-10 Protonated phosphoramidite

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1.12 The Polymerase Chain Reaction (PCR) Amplifies DNAs by Repeated Rounds of DNA R

eplication in Vitro

The Polymerase Chain Reaction (PCR) 聚合酶链反应 is a powerful method for amplifying particular segments of DNA,distinct from cloning and propagation within a host cell.

PCR uses the enzyme DNA polymerase that directs the synthesis of DNA from deoxynucleotide substrates on a single-stranded DNA template.

Steps:The DNA template is denatured by heating an

d annealed with synthetic oligonucleotide primer corresponding to the boundaries of the DNA sequence to be amplified.

Annealed with primers and used as a template for a fresh round of DNA synthesis.

DNA will have been synthesized by DNA polymerase.

Figure 20-11 Polymerase chain reaction

Repeated rounds of DNA duplication-whether carried out by cycles of cell division or cycles of DNA synthesis in vitro-amplify tiny samples of DAN into large quantities.

In cloning: often rely on a selective reagent or other device to locate the amplified sequence in an already existing library of clones.

In PCR: the selective reagent ,the pair of oligonucleotides, limits the qmplification process to the particular DNA sequence of interest from the beginning.

1.13 Nested Sets of DNA Fragments Reveal Nucleotide

Sequences

Sequenators( 序列分析仪 ) is a automatic sequencing machines.

To nested sets of DNA molecules created, one methods is chain-terminating nucleotides( 链终止法 )

Figure 20-12 Dideoxynucleotides used in DNA sequencing

Figure 20-13 Chain termination in the presence of dideoxynucleotides

Figure 20-14 DNA sequencing by the chain termination method

Figure 20-15 DNA sequencing gel

1.14 Shotgun Sequencing a Bacterial Genome

DNA was prepared from individual recombinant DNA colonies and separately sequenced on Sequenators using the dideoxy method is called shotgun.

This method might seem tedious,but it is considerably faster and less expensive than the techniques that were originally envisioned.

Figure 20-17

1.15 The Shotgun Strategy Permits a Partial Assembly of

LargeThe automated sequencing machines

are efficient.To determine the complete sequence

of the average human chromosome it is necessary to generate a large number of sequencing reads from many shot DNA fragmentsFigure20-16

Figure 20-16 Strategy for construction and sequencing of whole genome libraries

Recombinant DNA, containing a random portion of a human chromosome , can be rapidly isolated from bacterial plasmids and then quickly sequenced using the automated sequencing machines.

Contigs the short sequences from random shotgun DNAs into larger contiguous sequences.

Reads containing identical sequences are assumed to overlap and are joined to from larger contigs.Figure20-17

Figure 20-17 Contigs are linked by sequencing the ends of large DNA

fragments

1.16 The Paired-End Strategy Permits the Assmbly of Large

A major limitation to producing larger contigs is the occurrence of repetitive DNAs. Such sequences complicate the assembly process since random DNA fragments from unlinked regions of a chromosome or genome mighe appear to over due to the presence of the same repetitive DNA sequence. One method that is used to overcome this difficulty id called paired-end sequencing.

BAC(bacterial artificial chromosome): a special cloning vector to obtain paired-end sequence data from large DAN fragments that are at least 100kb in length.

The use of BACs often permits the assignment of multiple contigs into a single scaffold of several megabases.figure 20-17

1.17 Genome-Wide Analyses

Bioinformatics tools are required to identify genes and determine the genetic composition of complex.

Computer programs have been developed that identify potential protein coding genes through a variety of sequence criteria,including the occurrence of extended open-reading frames that are flanked by appropriate 5’and 3’splice sites.Figure 20-18

These methods not 100% accuracy.The most important method for calidating predic

ted protein coding genes and identifying those missed by current gent finder programs id the use of cDNA sequence dataFigure 20-18.cDNAs are generated by reverse transcriptionFigure 20-19 from mature mRNAs and hence represent bona fide exon sequences. The cDANs are used to generate EST (expressed sequence tag;a short sequence read from a large cDNA) date.

Figure 20-18 Gene finder methods:analysis of protein-coding regions in ciona

Figure 20-19 Synteny in the mouse and human chromosomes

1.18 Comparative Genome Analysis

Comparative analysis helps identify short exons, some located near the 5`end of the gene and the core promoter, that are often missed by gene prediction programs.

One of the striking findings of comparative genome analysis is the high degree of synteny, conservation in genetic linkage, between distantly related animals.

Figure 20-19 synteny in the mouse and human chromosomes

The most commonly used genome tool is BLAST (basic local alignment search tool).

3 steps in this process:you are asked which program you wish to use.To select a dataset.The results of the search are usually obtained i

n less than a minute.(the publicly available fly BLAST web site: www.f

ruitfly.org/blast/)

Figure 20-21 example of a BLAST search

The results of the search -----p672

The availability of whole genome swquences for an increasing number of animals is providing a rapidly expanding database for comparative genomics. The exon-intron nature of eukaryotic genes and the lack of strict sequence constraints in noncoding elements create formidable challenges to the identification of protein-coding sequences and regulatory elements by computational approaches. New and more effective tools of bioinformatics will be required to fully epxploit the treasure trove of information that is being generated by automated DAN sequencing.

PROTEINS

2.1 Specific Proteins Can Be Purified from Cell Extracts

The purification of individual proteins is critical to understanding their function.

The purification of a protein is designed to exploit its unique characteristics, including size, charge, shape, and in many instances,function.

2.2 Purification of a Protein Requires a Specific Assay

A DNA or RNA polymerase be assayed by adding the appropriate template and radioactive nucleotide precursor to a crude extract to label DNA.

Incorporation assay: are useful for monitoring the purification and function of many different enzymes catalyzing the synthesis of polymers.

2.3 Preparation of Cell Extracts Containing Active Proteins

The starting material for almost all protein purifications are extracts derived from cells.

Cells can be lysed by detergent, shearing forces, treatment with low ionic salt, or rapid changes in pressure, to weaken and break the membrane surrounding the cell to allow proteins to escape.

2.4 Proteins Can Be Separated from One Another Using Column Chromatography

Column chromatography( 柱层析法） The most common method for protein purification. Protein fractions are passed through glass columns filled with appropriately modified small acrylamide( 聚丙烯酰胺） or agarose （琼脂糖） beads. Separate proteins on the basis of their charge or size, respectively.

Figure 20-22 lon exchange and gel filtration chromatography

Lon exchange chromatography( 离子交换层析） the proteins are separated by their surface ionic charge using beads that are modified with either positively-charged chemical groups.

Gel filtration chromatography( 凝胶过滤层析 ) separates proteins on basis of size and shape.

Small proteins can enter all the pores and, can access more of the column and take longer to elute.

Large proteins can access less of the column and elute more rapidly..

2.5 Affinity Chromatography Can Facilitate More Rapid Protein Purification

Specific knowledge of a protein can frequently be exploited to purify a protein more rapidly.

Immunoaffinity chromatography( 免疫亲和层析 ) very common form of protein affinity chromatography.

An antibody that is specific for the target protein is attached to beads. Antibody will interact only with the intended target protein and allow all other proteins to pass through the beads . The bound protein can then be eluted from the column.

Adding short additional amino acid sequences to the beginning(N-terminus) or the end (C-terminus) of a target protein. Add to the modified proteins that assist in their purification.

Epitopes (a sequence of 7-10 amino acids recognized by an antibody) allow the modified protein to be purified using immunoaffinity purification and a heterologous antibody that is specific for the added epitope.

Immunoprecipitation ( 免疫沉淀 ) used to rapidly purify proteins or protein complexes from crude extracts.

Precipitation is achieved by attaching the antibody to the same type of bead used in column chromatography. Rapidly sink to the antibody.

2.6 Separation of Proteins on Polyacrylamide Gels

Sodium dodecylsulphate (SDS) a protein, treated with the strong ionic detergent, behaves as an unstructured polymer.

With mixtures of DNA and RNA, electrophoresis in the presence of SDS can be used to resolve mixtures of proteins according to the length of individual polypeptide chains. After electrophoresis, the proteins can be visualized with a stain

2.7 Antibodies Visualize Electrophoretically-Separated Proteins

Immunoblotting( 免疫印记法 ) steps: Electrophoretically separated proteins are transferr

ed and bound to a filter. Incubated in a solution of an antibody that had bee

n raised against an individual purified protein of interest. The antibody finds the corresponding protein on the filter to which it avidlybinds.

A chromogenic enzyme id used to visualize the filter-bound antibody.

2.8 Protein Molecules Can Be Directly Sequenced

Protein molecules can also be sequenced, the linear order of amino acids in a protein chain can be directly ditermined.

Two widely used methods for determining protein sequence:

Edman degradation : a chemical reaction in which the amino aci

d`s residues are sequentially released for the N-terminus of a polypeptide chain.

Figure 20-23 protein sequencing by edman degradation

Tandem mass spectrometry (MS/MS) : the mass of very small samples of a

material can be determined with great accuracy.

The principle is that material travels through the instrument in a manner that is sensitive to its mass/charge ratio.

Figure 20-24 analysis of the proteome by 2D electrophoresis and mass spectrometry

2.9 Proteomics

The availability of whole genome sequences in combination with analytic methods for protein separation and identification has ushered in the field of proteomics.