Techniques of Molecular Biology

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CHAPTER 20

• Techniquesof Molecular Biology

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Nucleic Acids

• Electrophoresis
• PCR
• Sequence

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Electrophoresis through a Gel Separates DNA and
RNA Molecular According To Size

• Pores in the gel matrix sieve the DNA molecules
  according to this volume.
• The large molecules move slower.
• After a given time, molecule of different size
  are separated because they have move different
  distances.
• Circularltlinearltsupercoiled

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Gel matrices

• Polyacrylamide narrow size range
• Agarose wide size range
• pulsed-field very large DNA(gt3050kb)

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Restriction Endonuclease Cleave DNA Molecular at
Particular Sites

• The large DNA must be broken into manageable
  fragments for analyzing.
• Restriction Endonuclease can recognize specific
  short (48bp) target sequence.

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Enzyme Sequence Cut Frequency
Sau3A1 EcoRI Not1 5-GATC-3 5-GAATTC-3 5-GCGGCCGC-3 0.25kb 4kb 65kb
Frequency1/4n ,where nthe number of bps in the
recognition sequence
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The hydrogen bonds between the 4bps between these
cut sites are easily broken to generate
5protruding ends of 4 nucleotides in length.
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DNA Hybridization Can Be Used to Identify
Specific DNA Molecules

• Hybridization the process of base-pairing
  between complementary single-stranded
  polynucleotide form two different sources.
• The probe is used to search mixtures of nucleic
  acids for molecules containing a complementary
  sequence.
• The probe DNA is labeled , and the mixture being
  probed is distributed as a library

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• Two methods for labeling DNA
• 1.synthesize new DNA in the presence of a
  labeled precursor.
• 2.add a label to the end of an intact DNA
  molecule.

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Hybridization Probes Can Identify
Electrophoretically-Separated DNAs and RNAs

• DNA fragments, generated by digestion of a DNA
  molecule by restriction enzyme ,are run out on an
  agarose gel.

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DNA Cloning

P C R
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Isolation of Specific Segment of DNA

• As molecular analysis requires the separation of
  specific segments of DNA , the ability to purify
  DNA is very important.
• Recombinant DNA can alter the expression of or
  generate DNAs.
• The techniques of DNA cloning and ampli-fication
  by PCR have become essential tools.

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DNA Cloning

• The constructing recombinant DNA and maintain
  them in cells is DNA cloning.
• The vector can provides the information necessary
  to propagate the cloned DNA.
• The insert DNA includes the target DNA is
  inserted within the vetor.

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Cloning DNA in Plasmid Vectors

• Vector DNAs have 3 characteristics
• 1.Contain an origin of replication which allow
  them to replicate in-dependently.
• 2.Contain a selectable mark that allows cells
  which have the vector to be identified.
• 3.Have single sites for several restriction
  enzyme.

See the film
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Vector DNA Can Be Introduce into Host Organisms
by Transformation

• Transformation is the process by which a host
  organism can take up DNA form its environment.
• Some bacteria have genetic competence to do this,
  while E.coli must be treated with Ca2.

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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 are the simplest libraries
  using insert DNA form total genomic DNA cleaved
  with a restriction enzyme.

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cDNA library

• Reverse transcription a special DNA polymerase
  that can make DNA from an RNA template.
• So RNA can be converted into dsDNA copies (cDNAs)

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

• Colony hybridization a labeled DNA probe is used
  to screen a library.
• The same type of positively-charged membrane
  filter used in the Southern and northern blotting
  techniques is again used to secure small amounts
  of DNA for probing.

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Chemically Synthesized Oligonucleotides

• Protonate phospho-amidine is the pre-cursor which
  are chemically protected molecule used in
  nucleotide addition.

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The Polymerase Chain Reaction (PCR) Amplifies
DNAs by Repeated Rounds of DNA replication in
Vitro
I want to see the movie!
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Sequence
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Nested Sets of DNA Fragments Reveal Nucleotide
Sequences

• Two methods are invented for creating nested sets
  of DNA molecules
• 1. They are radioactively labeled at their
  5termini and are then subjected to four
  different regimens of chemical treatment that
  cause them to break preferentially at Gs, Cs, Ts,
  or As.
• 2.Chain termination nucleotides Sequenators are
  based.

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Dideoxynucleotides used in DNA sequencingChain
termination in the presence of dideoxynucleotides
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DNA sequencing by the chain termination method
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DNA sequencing gel

• When we spike DNA synthesis reactions with ddCTP,
  ddATP, and ddTTP, then generated four nested sets
  of fragments.
• These fragments were resolved on a polyacrylamide
  gel .

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Shotgun Sequencing a Bacterial Genome

• The genome was randomly sheared into many random
  fragments with an average size of 1kb.
• These pieces were cloned into a plasmid
  recombinant DNA colonies.
• This is called shotgun sequencing.
• Random recombinant DNA colonies are picked,
  processed ,and sequenced.
• 10x sequence coverage means every nucleotide in
  the genome was sequenced ten times.

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The Shotgun Permits a Partial Assembly of Large
Genome Sequences
It is necessary to generate a large number of
sequencing reads from many short DNA fragment.
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• Recombinant DNA, containing a random portion of a
  human chromosome ,can be isolated from bacterial
  plasmids and then quickly sequenced using the
  automated sequencing machines, such as 384-column
  automated sequencing machine.

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The paired-End Strategy Permits the Assembly of
Large Genome Scaffolds
The use of BACs (bacterial artificial chromosome)
often permits the assignment of multiple contigs
into a single scaffold by virtue of sharing
several mesgabases.
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Genome-Wide Analyses
Gene finder methods analysis of protein-coding
regions in Ciona.
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Comparative Genome Analysis
One of the striking findings of Comparative
Genome Analysis is the high degree of Synteny ,
conservation in genetic linkage ,between
distantly related animals, such as human and
mice, showed in the figure.
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Comparison of a 34 kb region of the mouse and
human genomes
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Example of a BLAST search
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PROTEINS

• Purification from cell extract
• Separation through the column chromatography
• Separation on Polyacrylamide gels
• Immunoblotting
• Sequence directly
• proteomics

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Specific Proteins Can Be Purified from Cell
Extract

• The purification of proteins is a major part of
  understanding their function.
• Because the studies of the function of a protein
  in a complex mixture can often lead to
  ambiguities.
• It is designed to exploit its unique
  characteristics (size, charge, shape, and
  function )

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Purification of a Protein requires a Specific
Assay

• In the immunoblotting, antibody can detect
  proteins in the similar way of DNA hybridization.
• Incorporation assay a DNA or RNA polymerase can
  be assayed by adding the appropriate template and
  radioactive nucleotide precursor to crude extract.

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Preparation of Cell Extracts Containing Active
Protein

• Protein is not resilient to temperature, and
  denatured once released from the cell even in the
  moderate temperature.
• So most extracts preparation and protein
  purification is performed at 4?.
• For this, cells can be lysed by detergent,
  shearing forces, treatment with low ionic salt,
  and so on.

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Protein Can Be Separated from One Another Using
Column Chromatography

• Protein fractions are passed though glass columns
  filled with appropriately modified small
  acrylamide or agarose beads.
• Many ways columns can be used. And each one
  varies on the basis of different properties of
  proteins.
• The figure shows separation proteins by their
  charge or size.

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Other chromatography

• Ion exchange chromatography the proteins are
  separated by their surface ionic charge. Protein
  that interact weakly with the beads are release
  in a low salt buffer. By increasing the
  concentration of salt, protein with similar
  charge can be separated.
• Gel filtration chromatography it does by the
  basis of size and shape. The beads have a variety
  of different sized pores throughout.

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Affinity Chromatography Can Facilitate More Rapid
Protein Purification

• If a protein can binds ATP , it can be applied to
  a column of beads that are coupled to ATP.
• The protein you need will bind to the column
  while others will pass through.
• In the immunoaffinity Chromatography, the
  antibody is just like the ATP.

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Separation of Protein on Polyacrylamide Gels

• The SDS coat the proteins and impact on it a
  uniform negative charge, then the protein behaves
  as unstructured polymer.
• It can resolve mixtures of proteins according to
  the length of individual polypeptide chains.
• After electrophoresis, the protein can be
  visualized with a stain, such as Coomassie
  brilliant blue.

Wonderful movie
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Antibodies Visualize Electrophoretically-Separated
Proteins

• An protein is visualized amidst thousands of
  other protein by the immunoblotting.
• In it, Electrophoretically separated protein are
  transferred and bound to a filter.
• At last , a chromomeric enzyme is used to
  visualize the filter bound antibody

Just like it!
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Protein Molecule Can Be Directly Sequenced

• Edman degradation Cyclic degradation of
  peptides based on the reaction of
  phenylisothiocyanate (PITC) with the free amino
  group of the N-terminal residue such that amino
  acids are removed one at a time and identified as
  their phenylthiohydantoin derivatives.

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Edman degradation
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Edman degradation
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(No Transcript)
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Proteomics

• Based on 3 principal methods
• Two-dimensional gel electrophoresis for protein
  separation.
• Mass spectrometry for the precise determination
  of molecular weight and identify of a protein .
• Bioinformatics for assigning proteins and
  peptides to the predicted products of
  protein-coding sequence in the genome.

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Thank you!