1 chapter 20 techniques of molecular biology o4 级生物学基地班 林青青 200431060021

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Introduction• The methods depend upon,

and were developed from, an understanding of the properties of biological macromolecules themselves.

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Topic 1 nucleic acids

• Electrophoresis• Restriction• Hybridization• DNA Cloning and gene expression• PCR• Genome sequence & analysis • Comparative genome analysis

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1.Electrophoresis through a Gel separate DNA and

RNA molecules according to size

• Gel matrix an inert, jolly-like porous material that sieve

the DNA molecules according to its volumnDNA characteristics negatively charged, when subject to an elect

rical field, it migrates through the gel toward the positive pole

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Two types of normal gel matrices

• Polyacrylamide has high resolving capability but can s

eparate DNAs only over a narrow size range

• Agarose has less resolving power than polyacry

lamide but can separate from one another DNA molecules of up to tens, and even hundreds, of kilobases

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Fig 20-1: DNA separation by gel electrophoresis

http://a32.lehman.cuny.edu/molbio_course/agarose.htm

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Some fundamental steps of electrophoresis

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Whereas very long DNAs are unable to penetrate the pores in agarose

• DNA molecules above a certain size (30 to 50 kb) usually use pulsed-field electrophoresis to separate

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electrophoresis

DNA and RNA molecules are negatively charged, thus move in the gel matrix toward the positive pole (+)

Linear DNA molecules are separated according to size

The mobility of circular DNA molecules is affected by their topological structures. The mobility of the same molecular weight DNA molecule with different shapes is: supercoiled> linear> nicked or relaxed

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2.Restriction endonucleases cleave DNA molecules at partic

ular sites• endonucleases

– --To make large DNA molecules break into manageable fragments

• Restriction endonucleases: the nucleases that cleave DNA at particular sites by the recognition of specific sequences

• The target site recognized by endonucleases is usually palindromic

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To name a restriction endonuclease

e.g. EcoRI

Escherichia coli Species category

R13strain

the 1st such enzyme found

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• Endonucleases are used to make restriction map:– e.g. the combination of EcoRI + HindIII– Allows different regions of one molecule to

be isolate and a given molecule to be identified

– A given molecule will generate a characteristic series of patterns when digested with a set of different enzymes

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Different enzymes recognize their specific target sites with different frequency

• EcoRI Recognize hexameric sequence: 4-6

• Sau3A1 Recognize terameric sequence: 4-4

• Thus Sau3A1 cuts the same DNA molecule more frequently

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Recognition sequences and cut sites of various endonucleases

blunt ends

sticky ends

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The 5’ protruding ends of are said to be “sticky” because they readily anneal

through base-pairing to DNA molecules cut with the

same enzyme

• Reanneal with its complementary strand or other strands with the same cut

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3.DNA hydridization can be used to identify specific

DNA molecules• Hybridization: the process of base-pairing between co

mplementary single-stranded polynucleotides from two different sources

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probeNotes• Probe is a specific DNA or RNA fragment which can

bind with the sample DNA or RNA for detection. ATCCGATCG--------

• Source of probe synthesized, cloning genomic DNA or cDNA, as well a

s RNA. • Probe must be labeled before hybridization. radioactive αorγ32P nonradioactive biotin, digoxigenin, fluorescent dy

e• In a single stranded form for hybridization

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There are two basic mothods for labeling DNA:

• Synthesizing new DNA in the presence of a labeled precursor

• Adding a label to the end of an intact DNA molecule

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Labeling of DNA or RNA probes

• radioactive labeling: display and/or magnify the signals by radioactivity

• Non-radioactive labeling: display and/or magnify the signals by antigen labeling – antibody binding – enzyme binding - substrate application (signal release)

• End labeling: put the labels at the ends• Uniform labeling: put the labels

internally

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Uniformly labeling of DNA/RNA

• Nick translation: • DNase I to introduce random nicks DNA pol

I to remove dNMPs from 3’ to 5’ and add new dNMP including labeled nucleotide at the 3’ ends

• Hexanucleotide primered labeling: • Denature DNA add random hexanucleot

ide primers and DNA pol synthesis of new strand incorporating labeled nucleotide

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Ways of Molecular Hybridization

A. Transfer blotting ( 转移印迹 ) Southern blotting Northern blotting Western blotting Eastern blotting

B. Dot blotting & Slot blotting ( 点印迹 , 狭缝印迹 )

C. In situ hybridization ( 原位杂交 )

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Southern and Northern blotting

1. Genomic DNA preparation RNA preparation

2. Restriction digestion -3. Denature with alkali - 4. Agarose gel electrophoresis 5. DNA blotting/transfer and fixation RNA6. Probe labeling 7. Hybridization (temperature) 8. Signal detection (X-ray film or antibody)

DNA on blot RNA on blot

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Blot type

Target

Probe Applications

Southern DNA DNA or RNA

mapping genomic clones

estimating gene numbers

Northern RNA DNA or RNA

RNA sizes, expressionabundance,

and

Western Protein

Antibodies

protein size, abundance

Characteristics of transfer bloting

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Southern blotting• It is first proposed by Dr. Edwin Southern in

Edinburgh University in 1975, and term “Southern blotting” is named for him.

• Major steps: electrophoresis transfer blotting molecular hybridization

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Southern analysis

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DNA 样品

DNA 探针

变性

X-ray 片

限制性内切酶消化

琼脂糖凝胶电泳

转移印迹

胶 膜

两部分工作标记

杂交

暴光

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Northern blot hybriodization• Can be used to identify a particular mRNAs• The protocol is fairly similar to that describe

for southern blotting except that mRNA are not needed to be digested with any enzymes

• An experimenter might carry out northern blot hybridization to ascertain the amount of a particular mRNA present in a sample rather than its size

• Moreover, northern blot hybridization might be carried out to compare the relative levels of a particular transcript between tissues of an organism

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4. DNA cloning

• DNA cloning: the ability to construct recombinant DNA

molecules and maintain them in cellsThis 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

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5. PCR• The polymerase chain reaction (PCR) a

mplifies DNAs by repeated rounds of DNA replication in vitro

• PCR is used to amplify a sequence of DNA u

sing a pair of primers each complementary to one end of the DNA target sequence

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Cloning DNA in plasmid vectors

Vector DNAs typically have three characteristics:1. An origin of replication that allow them to re

plicate independently of the chromosome of the host

2. A selectable marker that allows cells that contain the vector to be readily identified

3. Single sites for one or more restriction enzymes that allow DNA fragments to be inserted at a defined point within an otherwise intact vector

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Vector DNA can be introduced into host

organisms by transformation

• Transformation the process by which a host

organism can take up DNA from its environment

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• Genetic competence• An antibiotic to which the plasmid

imparts resistance is then used to select transformants that have acquired the plasmid

• Transformation generally is a relatively inefficient process

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Libraries of DNA molecules can be created

by cloningGenerate a specific clone• If the starting donor DNA is simple----restriction enzyme & gel electrophoresis

• If the starting DNA is more complex----clone the whole population of fragment

& separate the individual clones

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DNA libraryA population of identical vectors that e

ach contains a different DNA insert Genomic library (the simplest)

cDNA library

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Polymerase Chain Reaction

• The PCR consists of three defined sets of temperatures and times termed steps:

• (1) denaturing, (2) annealing, (3) extension.

Denaturing 940C 45 Sec

Annealing 550C-630C 30 Sec 30 cycles

Extension 720C 45 Sec

Annealing temperature: Ta=Tm-5 C

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(1) Template

•Any source of DNA that provides one or more target molecules can in principle be used as a template for PCR

•Whatever the source of template DNA, PCR can only be applied if some sequence information is known so that primers can be designed.

J3 Polymerase chain reaction

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(2) Primers• PCR primers need to be about 18 to 30 nt lo

ng and have similar G+C contents so that they anneal to their complementary sequences at similar temperatures.They are designed to anneal on opposite strands of the target sequence.

• Tm=2(a+t)+4(g+c): determine annealing temperature. If the primer is 18-30 nt, annealing temperature can be Tm5oC

J3 Polymerase chain reaction

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(3) A pair of primers

The key to the PCR lies in the design of the primers:

A.20-30 bp in length with each complementary

to the 3’ side in a strand of target DNA. B. not self-complementary C. not consecutive 4 same bases (AAAA) D. proper GC content (40-60%) primer sequence from Genbank, designed by software

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(4) DNA polymerases (Taq polymerases)

It is thermostable, temperature optimum is 720C and active when the temperature over 960C. It was first isolated from the thermophilic

bacterium （ Thermus aquaticus ） found in hot springs.

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5’3’

5’3’

5’ 3’

5’ 3’

5’

5’

3’

3’

5’ 3’

5’3’

5’3’

5’3’

5’3’

5’ 3’

5’ 3’

5’ 3’

5’3’

5’ 3’

5’3’

5’ 3’

5’5’ 3’3’

5’

5’3’

5’ 3’

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3’

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Denaturation

Annealing

Extension

Cycle 1

Cycle 2

Cycle 3

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• Rate of PCR 2n

InitialDNA

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Number of DNA molecules

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PCR optimization

I.Reverse transcriptase-PCR

II.Nested PCR

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Similarity and difference between DNA cloning and

PCR• Similarity: repeated rounds of DNA duplication

• Difference: DNA cloning --- rely on a selective reagent or other

device to locate the amplified sequence in an already existing library of clones

PCR --- the selective reagent, the pair of oligonucleotides, limits the amplification process to the particular DNA sequence of interest from the beginning

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5.Genome sequence & analysis

• Nested sets of DNA fragments reveal nucleotide sequences

• Shotgun sequencing a bacterial genome

• The shotgun strategy permits a partial assembly of large genome sequences

• The paired-end strategy permits the assembly of large genome scaffolds

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SequencingSequencing

Two ways for sequencing:– 1. DNA molecules

(radioactively labeled at 5’ termini) are subjected to 4 regiments to be broken preferentially at Gs, Cs, Ts, As, separately.

– 2. chain-termination method

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chain-termination method

• ddNTPs are chain-terminating nucleotides: the synthesis of a DNA strand stops when a ddNTP is added to the 3’ end

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The absence of 3’-hydroxyl lead to the inefficiency of the nucleophilic attack on the next incoming substrate molecule

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DNA synthesis aborts at a frequency of 1/100 every time the polymerase meets a ddGTP

Tell from the gel the position of each G

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Shotgun sequencing a bacterial genome

• The bacterium Hemophilus influenzae was the first free-living organism to have a complete genome sequence and assembly

• This organism is chosen as its genome is small (1.8Mb) and compact

• Its whole genome was sheared into many random fragments with an average length of 1kb.

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• This pieces are cloned into a plasmid vector. And these clones are sequenced respectively.

• All these sequence information are loaded into the computer. The powerful program will assemble the random DNA fragment based on containing matching sequence, forming a single continuous assemble, called a contig.

• To ensure every nucleotide in the genome was captured in the final genome assemble, 30000~40000 clones are needed, which is ten times larger as the genome. This is called 10×sequence coverage.

• This method might seem tedious, but it’s much faster and cheaper than the digestion-mapping-sequencing method. As the computer is much faster at assembling sequence than the time required to map the chromosome.

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The shotgun strategy permits a partial assembly of

large genome sequence• Recombinant DNA can be rapidly isolated f

rom bacterial plasmids and then quickly using the automated sequencing machines

• Sophisticated computer programs have been developed that assemble the short sequence from random shotgun DNAs into large contiguous sequence called contigs

54Fig 20-16

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The paired-end strategy permits the assembly of large genome

scaffolds• The main limitation to producing large conti

gs is the occurrence of repetitive sequence. • To solve this problem, paired-end sequencin

g is developed.• The same genomic DNA is also used to prod

uce recombinant libraries composed of large fragments between 3~100kb.

• The end of each clone can be sequenced easily, and these larger clones can firstly assemble together.

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Genome-wide analyses• The purpose of this analysis is to predict the coding

sequence and other functional sequence in the genome• For animal genomes, a variety of bioinformatics tools

are required to identify genes and other functional fragments. But the accuracy is low

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• The most important method for validating protein coding regions and identify those those missed by current current gene finder program is the use of cDNA sequence data.

• The mRNAs are firstly reverse transcript into cDNA, and these cDNA, both full length and partial, are sequenced using shortgun method. These sequence are used to generate EST (expressed sequence tag) database. And these ESTs are aligned onto genomic scaffolds to help us identify genes.

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6. Comparative genome analysis

• The comparison of different animal genomes permits a direct assessment of changes in gene structure and sequence that have arisen during evolution

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

• The most commonly used genome tool is BLAST

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Topic 2proteins

• Purification• Separation• Sequencing• proteomics

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1. purification (1) specific proteins can be purified from

cell extracts• The purification of individual proteins is

critical to understanding their function• Each protein has unique properties that

make its purification somewhat different• The purification of a protein is designed

to exploit its unique characteristics, including size, charge, and in many instances, function

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(2) Purification of a protein require a specific assay

• To purify a protein requires that you have an assay that is unique to that protein

• In many instance, it is more convenient to use a more direct measure for the function of the protein

• Incorporation assay: are useful for monitoring the purification

and function of many different enzymes

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(3) Preparation of cell extracts containing active proteins

• Most extract preparation and protein purification is performed at 4 。 C

• Cell extracts are prepared in a number of different ways:

Exa: cells can be lysed by detergent, shearing forces, treatment with low ionic salt, or rapid changes in pressure

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2.Separation (1) proteins can be separated from one

another using column chromatography

• Column chromatography in this approach to protein purification, pr

otein fractions are passed through glass column filled with appropriately modified small acrylamide or agarose beads.

• There are various ways columns can be used to separate proteins

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Ion exchange chromatography

• The proteins are separated according to their surface charge.

• The beads are modified with either negative-charged or positive-charged chemical groups.

• Proteins bind more strongly requires more salt to be eluted.

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Gel filtration chromatography • This technique

separate the proteins on the bases of size and shape.

• The beads for it have a variety of different sized pores throughout.

• Small proteins can enter all of the pores, and take longer to elute; but large proteins pass quickly.

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(2) Affinity chromatography can facilitate more rapid protein

purification If we firstly know our target protein can

specifically interact with something else, we can bind this “something else” to the column and only our target protein bind to the column.

This method is called affinity chromatography.

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AffinityAffinitychromatographychromatography

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Immunoaffinity chromatography• An antibody that is specific for the

target is attached to the bead, and ideally only the target protein can bind to the column.

• However, sometimes the binding is too tight to elute our target protein, unless it is denatured. But the denatured protein is useless.

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• Sometimes tags (epitopes) can be added to the N- or C- terminal of the protein, using molecular cloning method.

• This procedure allows the modified proteins to be purified using immunoaffinity purification and a heterologous antibody to the tag.

• Importantly, the binding affinity can change according to the condition. e.g. the concentration of the Ca2+ in the solution.

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immunoprecipitation• We attach the antibody to the bead,

and use it to precipitate a specific protein from a crude cell extract.

• It’s a useful method to detect what proteins or other molecules are associated with the target protein.

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(3) Separation of proteins on polyacrylamide

• Proteins have neither a uniform negative nor a uniform secondary structure

• if, however, a protein is treated with the strong ionic detergent sodium dodecyl sulphate (SDS) and a reducing agent, such as mercaptoethanol, the secondary, tertiary, and quarternary structure is usually eliminated

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SDS ions coat the polypeptide chain and thereby impart on it a uniform negative chargeMercaptoethanol reduces disulphide bonds and thereby disrupts intramolecular and intramolecular disulphide bridges formed between cysteine residues Thus, as is the case 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 chainsAfter electrophoresis, the proteins can be visualized with a stain,such as Coomassie brilliant blue, that binds to protein

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(4) Antibodies visualize electrophoretically-separated protein

• The electrophoretically separated proteins are transferred to a filter.

• And this filter is then incubate in a solution of an antibody to our interested protein.

• Finally, a chromogenic enzyme is used to visualized the filter-bound antibody

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3. sequencingProtein molecules can be directly sequenced

• Due to the vast resource of complete or nearly complete genome, the determination of even a small stretch of protein sequence is sufficient to identify the gene.

Two sequence method: Edman degradation & Tandem mass spectrometry(MS/MS).

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Edman degradation• It’s a chemical

reaction in which the amino acid’s residues are sequentially release for the N-terminus of a polypeptide chain

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• Step 1: modify the N-terminal amino with PITC, which can only react with the free α-amino group.

• Step 2: cleave off the N-terminal by acid treatment, but the rest of the polypeptide remains intact.

• Step 3: identify the released amino acids by High Performance Liquid Chromatography (HPLC).

The whole process can be carried out in an automatic protein sequencer.

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Tandem mass spectrometry

• MS is a method in which the mass of very small samples of a material can be determined.

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• Step 1: digest your target protein into short peptide.

• Step 2: subject the mixture of the peptide to MS, and each individual peptide will be separate.

• Step 3: capture the individual peptide and fragmented into all the component peptide.

• Step 4: determine the mass of each component peptide.

• Step 5: Deconvolution of these data and the sequence will be revealed.

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4. proteomics• Proteomics is concerted with the

identification of the full set of proteins produced by a cell or tissue under a particular set of conditions, their relative abundance, and their interacting partner proteins

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Proteomics is based on three principal methods:

1. two-dimensional gel electrophoresis for protein separation

2. Mass spectrometry for the precise determination of the molecular weight and identity of a protein

3. Bioinformatics for assigning proteins and peptides to the predicted products of protein-coding sequences in the genome

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The The EndEnd