Techniques of molecular biology

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Chapter 20

• Techniques of molecular biology

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Intronduction

• The living cell is an extraordinary complicated
  entity, with thousands of macromolecules in it.
• So is important to separate individual
  macromolecules from the myriad mixtures, to
  dissect the genome into manageable size segment
  for manipulation and analysis

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Intronduction

• Recently ,it has become possible to apply
  molecular approaches to the large-scale analysis
  of the full complement of RNAs and proteins in
  the cell and to determine the sequence of an
  entire genome.

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Intronduction

• Two parts of this chapter
• Part 1
• techniques for the manipulation and
  characterization of nucleic acid
• Part 2
• techniques for the isolation and analysis of
  protein

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Part 1 Nucleic acids

• Separate DNAs and RNAs
• Identify specific DNA molecules
• Isolation of specific DNAs molecules
• DNA cloning
• DNA sequencing

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1.1.2 Separate DNAs and RNAs

• Gel electrophoresis
• separate DNA according to the length, shape,
  topological properties
• Why
• How
• When

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Why

• DNA is negatively charged, so they will migrate
  to the positive pole once put in an electronic
  field
• The rates they travel through the gel are
  different for their different length. The longer
  the DNA is ,the slower its rate will be
• After the electrophoresis the DNA can be
  visualized by staining the gel with fluorescent
  dyes, such as ethidium

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How

• Constant electrophoresis
• Polyacrylamide
• high resolving capability
• narrow size range
• Agarose
• less resolving capability
• large NDA and their diffrences

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How

• Pulsed field
• Very long DNAs are unable to penetrate even with
  agarose.
• Pulsed field is used to solve this problem.

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When

• Electrophoresis is used to separate
• DNAs of different length
• DNAs of different shape
• eg.linear and circular
• DNA of different topological properties
• eg. supercoiled and less supercoiled DNA
• RNA
• single strand
• secondary and tertiary structure

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1.1.2 Restriction endonuclease cleavage

• Restriction endonuclease can find the specific
  site of DNA and cut it to separate the DNA from
  the genome to form manageable fragments
• Eg . EcoR1

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• restriction enzymes differ in
• recognition sequence
• cut frequency(1/4n)
• cut site

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1.2 Identify specific DNA molecules

• DNA hybridization
• Base-pair between two single -stranded
  polynucleotide from different sources is called
  hybridization
• Probe
• The defined sequence is called probe, either
  a purified fragment or a chemically synthesized.
  The probe must be labeled so that it can be
  readily located.

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Two basic methods to label a probe

• 1.synthesis of a new DNA in the presence of the
  labeled precursor.
• Use PCR with a labeled precursor ,or hybridize
  short random hexameric oligonucleotides to DNA
  and allow a DNA polymerase to extend them
• The label precursors are most commonly nucleotide
  modified with a fluorescent moiety or
  radioactivity atoms.
• The DNA labeled with fluorescent precursors can
  be detected by radiating the sample with UV
• DNA Labeled with radioactive atoms can be
  detected by exposed to X-ray film or by
  photomultipliers
• 2.adding a label to the end of an intact DNA
  molecules

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Usage

• Southern blot
• restriction enzyme cut a specific fragments
• electrophoresis of these fragment
• transfer to a filter and detect a specific
  sequence with a probe homologous to it
• This can detect the amount of the specific
  sequence

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Usage

• Northern blot
• To monitor the amount of a specific mRNA.
• This a reflection of the expressing level of a
  gene.

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1.4 Isolation of specific DNAs molecules

• Much molecular analysis requires the separation
  of specific segment of DNA from much larger DNA
  molecules and their amplification. This is
  important to
• DNA analysis
• DNA sequencing
• DNA manipulation

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1.4.1 DNA cloing

• DNA cloning
• The ability to construct recombinant DNA
  molecules and maintain them in the cell is
  called.
• Vector
• provide the information necessary to propagate
  the cloned DNA
• Inserted DNA
• inserted into the vector and include the DNA of
  interest

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1.4.2 Cloning DNA in plasmid vector

• Vector DNA has three characteristics
• origin of replication to allow them to replicate
  independently
• selectable marker to allow cells contain the
  vector be identified
• single site for one or more restriction enzyme
  to allow DNA fragments to be inserted into it

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Plasmid

• From bacteria ,single-cell eukaryotes
• Propagate independently
• Carry gene encode resistance to antibiotics
• Carry useful restriction site
• Some drive the expression of gene( express
  vector)

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Vector is carried into the host cell

• Transformation
• a host organism can take up DNA from the
  environment
• Genetic competence
• only a bacteria of genetic competence can
  execute transformation
• E.coli can be reddened competent to take up
  DNA by treatment with calcium ions. An antibiotic
  to with the plasmid impacts resistance is the
  use to select transformations.

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1.4.3 Libraries of DNA molecules can be created
by cloning

• For complex starting DNA
• A population of identical vector that each
  contain a different DNA inserts
• Restriction enzyme give a desired average insert
  size
• Genomic libraries DNA libraries for a whole
  genome

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cDNA library

• A mRNA is converted to DNA strands
• mRNA DNA
• Hybridization can be used to identify a specific
  clone in a DNA library, which is called cloning
  hybridization.
• Positively charged membrane filter is used in
  this procession

Reverse transcription
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1.4.4 Chemical synthesized oligonucleotide

• Short ,custom-designed segment of DNA knows as
  oligonucleotide.
• Solid supports using machines automate the
  process
• The protected resides are called phosphoamidines
• 30 bp is of enough accuracy, however, longer DNA
  synthesis final product is less uniform due to
  the inherent fails
• Usage
• site-directed mutagenesis
• probe in hybridization
• primer in PCR

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1.4.5 The Polymerase Chain Reaction( PCR)

• Acquire
• DNA polymerase
• oligonucleotide
• single strand template
• Steps
• 1.heat denature single strands
• 2.add primers primer-template junction
• 3.add DNA polymerase DNA synthesis
• 4.goto Step 1

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1.5 DNA sequencing

• Nested set of DNA fragments
• Two methods
• 1 DNA molecules are redioactively labeled at
  their 5termis, and then subjected to four
  different regions of chemical treatment that
  cause them to break preferentially.
• 2 chain-termination nucleotides
• 2-,3 didexynucleotide (ddNTPs)
• ddNTP2-dexoy-NTP1100

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1.5.1 Shotgun sequencing

• For large genome
• DNA was prepared from a bacteria genome
  individual recombinant DNA clone and separately
  sequence on a sequenators.
• 10Xsequence coverage
• fast and less expensive than systematically
  sequencing every defined restriction DNA fragment
  on the physical map of that bacteria chromosome
  
• setbacks
• once a single site is not correctly
  identified ,the whole genome may be wrong
• contigs are linked by sequencing the end of
  large DNA fragments.

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1.5.2 Genome-wide Analysis
Bacteria and single eukaryotes Straightformed,
effective Key challenge is in identification the
function of the genes
Animal genome Complex exon-intron structure No
100 accuracy to final exon Fail to identify
promoters
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1.5.3 Comparative genome analysis

• High degree of synteny
• BLATS( basic local alignment search tool ) is
  used to find region of similarity between
  different protein coding gene, search the genome
  to find query sequence

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Part 2 Proteins

• 2.1.Purification
• require a specific array, based on any of their
  features ,including weight, shape, charges they
  carrying, other specific features.
• incorporation assays
• immunoblotting( specific interaction between
  Ab and Ag)
• a specific DNA for an DNA binding protein

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Steps

• Preparation of cell extracts contain active
  proteins .
• To protect the activation of the proteins
• 1.fitting temperature 4 oC
• 2.fitting ionic salt

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2.2 Separate protein using column chromatography

• Many way columns can be used
• 1 Icon exchange chromatography
• separate proteins by their surface ionic charge
  
• 2 gel filtration chromatography
• separate proteins on the basic of size and shape

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• Affinity chromatography
• rapid protein purification
• ATP -----beads ATP binding proteins
• immunoaffinity chromatography
• Ab-----beads Ag
• Ag------Beads Ab
• modified protein

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Polycacylamide gel

• Sodium Dodecyl sulphate ( SDS)
• The mercartoethanol ,secondary, tertiary,
  quarternary structure is usually eliminated. The
  proteins are separated on the basic of weight.
• visualize the proteins
• Coomassie brilliant blue
• immunoblotting

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2.3 Protein molecule can be directly equenced

• Two widely used ways
• Edman degradation
• chemical reaction, in which the amino acid
  residues are sequentially released from the
  N-terminus of a polypeptide chain
• Tanden mas spectrometry( MS/MS)
• principle material travels through the
  instrument in a manner that is sensate to its
  mass/charge ratio
• protein of interest must be digested into short
  pettides.

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2.4 Proteomics

• Proteomics is concerned with the identification
  of the full set of protein produced by a cell or
  tissue under a particular set of condition ,that
  relative abundance ,and their interacting partner
  proteins.

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• Proteomics base on three principal method
• 1 Two-dimensional gel electrophoresis
• 2 mass spectronetry for precise determination
• 3 bioinformatics to assigning proteins and
  peptides and to the predicted products of protein
  coding sequences

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