Techniques of Molecular Biology

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Chapter 20
Techniques of Molecular
Biology
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OUTLINE

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

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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.

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

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• 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.

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• 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

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

• 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.

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• Gel electrophoresis (????) 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)??

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• DNA molecules are flexible and occupy an
  effective volume pores in the gel matrix sieve
  the DNA molecules according to this volumelarge
  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.

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Figure 20-1 DNA separation by gel electrophoresis
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• After electrophoresis is complete
• DNA molecules can be visualized by staining the
  gel with fluorescent dyes(such as ethidium????)

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• Two alternative kinds of gel matrices
• Polyacrylamide(?????)has high resolving
  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(???)has less resolving power than
  polyacrylamide but can separate from one another
  DNA molecules of up to tens,and even hundreds,of
  kilobases.

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• Pulsed-field gel electrophoresis(????????)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

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• 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.

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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.

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Figure 6-26 schematic of electrophoretic
separation of DNA topoisomers.
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1.2 Restriction Endonucleases Cleave DNA
Molecules at Particular Sites

• Restriction endonucleasesnucleases 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.

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• 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)

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Figure 20-3 Digestion of a DNA fragment with
endonuclease EcoRI
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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
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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
  5or 3 overhanging ends can be generated.

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Figure 20-5 Cleavage of an EcoRI site
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1.3 DNA Hybridization Can Be Used to Identify
Specific DNA Molecules

• Hybridization(??)
• The capacity of denatured to reanneal allows for
  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.

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• Probe(??)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.

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

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1.4 Hybridization Probes Can Identify
Electrophoretically Separated DNAs and RNAs

• Southern blot hybridization(????)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.

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• Autoradiogram(?????)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 .

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Figure 20-6 A Southern Blot
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• 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.

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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.

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1.6 DNA Cloning

• DNA cloningto 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

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1.7 Cloning DNA in Plasmid Vectors

• The DNA fragment must be inserted within that
  second DNA molecule to be replicated in a host
  organism.
• Vector DNAs typically have three characteristics
• They contain an origin of replication that allows
  them 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.

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• Plasmids(??)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,carry a
  selectable marker.
• Present in multiple copies per cell. This
  increases the amount of DNA that can be isolated
  from a population.

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• 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.

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Figure 20-7 Cloning in a plasmid vector
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• 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 genes to
  assess their function.
• Used to produce large amounts of a protein for
  purification

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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.

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• 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.

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1.9 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.Figure 20-8
• Genomic libraries(?????) derived from total
  genomic DNA cleaved with a restriction enzyme.

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• Reverse transcription(???) 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

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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.

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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

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• 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.

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Figure 20-10 Protonated phosphoramidite

• ?
• ?

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1.12 The Polymerase Chain Reaction (PCR)
Amplifies DNAs by Repeated Rounds of DNA
Replication in Vitro

• The Polymerase Chain Reaction (PCR) ?????? is a
  powerful method for amplifying particular
  segments of DNA,distinct from cloning and
  propagation within a host cell.

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

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• Steps
• The DNA template is denatured by heating and
  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.

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• Figure 20-11 Polymerase chain reaction

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• 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.

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1.13 Nested Sets of DNA Fragments Reveal
Nucleotide Sequences

• Sequenators(?????) is a automatic sequencing
  machines.
• To nested sets of DNA molecules created, one
  methods is chain-terminating nucleotides(????)

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Figure 20-12 Dideoxynucleotides used in DNA
sequencing
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Figure 20-13 Chain termination in the presence of
dideoxynucleotides
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Figure 20-14 DNA sequencing by the chain
termination method
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Figure 20-15 DNA sequencing gel
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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

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1.15 The Shotgun Strategy Permits a Partial
Assembly of Large

• The 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

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

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

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• 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

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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 5and 3splice
  sites.Figure 20-18

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• These methods not 100 accuracy.
• The most important method for calidating
  predicted 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 taga short
  sequence read from a large cDNA) date.

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Figure 20-18 Gene finder methodsanalysis of
protein-coding regions in ciona
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Figure 20-19 Synteny in the mouse and human
chromosomes
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1.18 Comparative Genome Analysis

• Comparative analysis helps identify short exons,
  some located near the 5end 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.

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Figure 20-19 synteny in the mouse and human
chromosomes
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• 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 in
  less than a minute.
• (the publicly available fly BLAST web site
  www.fruitfly.org/blast/)

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Figure 20-21 example of a BLAST search
The results of the search -----p672
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• 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.

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PROTEINS
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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.

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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.

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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.

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2.4 Proteins Can Be Separated from One Another
Using Column Chromatography

• Column chromatography(????)The most common method
  for protein purification. Protein fractions are
  passed through glass columns filled with
  appropriately modified small acrylamide(?????) or
  agarose(???) beads. Separate proteins on the
  basis of their charge or size, respectively.

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Figure 20-22 lon exchange and gel filtration
chromatography
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• Lon exchange chromatography(??????)the proteins
  are separated by their surface ionic charge using
  beads that are modified with either
  positively-charged chemical groups.

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• Gel filtration chromatography(??????) 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..

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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.

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• Immunoaffinity chromatography(??????) 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.

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• 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.

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• Immunoprecipitation (????) 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.

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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

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2.7 Antibodies Visualize Electrophoretically-Separ
ated Proteins

• Immunoblotting(?????) steps
• Electrophoretically separated proteins are
  transferred and bound to a filter.
• Incubated in a solution of an antibody that had
  been 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.

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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.

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• Two widely used methods for determining protein
  sequence
• Edman degradation
• a chemical reaction in which the amino acids
  residues are sequentially released for the
  N-terminus of a polypeptide chain.

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• Figure 20-23 protein sequencing by edman
  degradation

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• 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.

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

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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.