Techniques of Molecular Biology

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Chapter 20

• Techniques of Molecular Biology

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The methods of molecular biology depend upon and
were developed from an understanding of the
properties of biological macromolecules
themselves.
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Part I NUCLEIC ACID
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NUCLEIC ACIDS
DNA and RNA separation by gel electrophoresis

• Principle Linear DNA molecules migrate through
  the gel toward the positive pole with different
  rates when subject to an electrical field.
• The DNA molecules can be visualized by staining
  the gel with fluorescent dyes, such as ethidium.

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NUCLEIC ACIDS

• Two matrices polyacrylamide and agarose.
  Plyacrylamide has more resoving power.
• Pulsed-field gel electrophoresis for long DNAs
  (up to several Mb in length).

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According to RNA it is similar, however RNA
sample should be treated with reagents ,e.g.
glyoxal to prevent the formation of base pairs.
NUCLEIC ACIDS
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Restriction Endonuleases Cleaves DNA Molecules at
Particular Sites
NUCLEIC ACIDS

• Restriction enzymes recognize short target
  sequences and cut at a defined position within
  those sequences.
• They can generate different ends flush ends and
  staggered ends.
• We use them to break large DNA into manageable
  fragments.

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NUCLEIC ACIDS
Recognition sequences and cut sites of various
endonucleases
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• How we name them??
• Take EcoRI for example
• Eco E. coli
• I the first one

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Hybridization probes can identify
electrophoretically separated DNA and RNA
NUCLEIC ACIDS

• Southern blot named after Edward Southern
• DNA fragments, generated by digestion of a DNA
  molecule by a restriction enzyme, are run out on
  an agarose gel.
• Once stained, a pattern of fragments is seen.
• When transferred to a filter and probed with a
  DNA fragment homologous to just one sequence in
  the digested molecule, a single band is seen,
  corresponding to the position on the gel of the
  fragment containing that sequence.

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NUCLEIC ACIDS
One example of southern blot
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DNA Cloning
NUCLEIC ACIDS

• Some termsDNA cloning vector insert
  DNAlibrary a population of identical vectors
  that each contains a different DNA insert.

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NUCLEIC ACIDS

• Characteristics of vector DNAs1.an origin of
  replication2.a selectable marker3.sigle sites
  for one or more restriction enzymes.

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How to clone DNA in plasmid vectors
NUCLEIC ACIDS

• A fragment of DNA , generated by cleavage with a
  certain restriction enzyme, is inserted into the
  plasmid vector linearized by the same enzyme.
• The recombinant plasmid is introduced int o
  bacteria by transformation.
• Cells containing the plasmid can be selected by
  growth on the antibiotic to which the plasmid
  confers resistance.

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Construction of a genomic DNA library
NUCLEIC ACIDS

• Genomic DNA and vector DNA, digested with the
  same restriction enzyme, are incubated together
  with ligase
• The resulting pool or library of hybrid vectors
  is then introduced into E. coli, and the cells
  are plated onto a filter placed over agar medium.
• The filter is removed from the plate and prepared
  for hybridization.

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NUCLEIC ACIDS
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Construction of a cDNA library
NUCLEIC ACIDS

• Isolate mRNA
• use reverse transcriptase to synthesize
  complementary DNA strand from mRNA, then use DNA
  Pol I to synthesize double stranded DNA. Clone
  these cDNAs into appropriate vector (usually
  plasmid or phage)
• Use Oligo dT primer to hybridize to polyA tail of
  mRNA. Primer used by reverse transcriptase for
  extension.
• Reverse transcriptase is a DNA polymerase which
  uses RNA as a template to synthesize
  complementary DNA. Cloned from RNA viruses.

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We should note that
NUCLEIC ACIDS

• No introns cloned, nor regulatory sequences
• Genes cloned in this method are only those that
  were expressed in the particular tissue mRNA was
  isolated from.

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NUCLEIC ACIDS
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NUCLEIC ACIDS

• After having constructed a DNA library,
  whether genomic or cDNA, we can use probes to
  find specific clones we are interested in.

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Site-directed mutagenesis
NUCLEIC ACIDS

• Using site-directed mutagenesis the
  information in the genetic material can be
  changed. A synthetic DNA fragment is used as a
  tool for changing one particular code word in the
  DNA molecule. This reprogrammed DNA molecule can
  direct the synthesis of a protein with an
  exchanged amino acid.

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Polymerase Chain Reaction
NUCLEIC ACIDS
The Royal Swedish Academy of Sciences awards
1993s Nobel Prize in Chemistry to
For more, click http//nobelprize.org
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NUCLEIC ACIDS

• for contributions to the developments of methods
  within DNA-based chemistry
• for his invention of the polymerase chain
  reaction (PCR) method
• for his fundamental contributions to the
  establishment of oligonucleotide-based,
  site-directed mutagenesis and its development for
  protein studies

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Lets look into it in more details
NUCLEIC ACIDS

• Denaturation at 94? the double strand melts
  open to single stranded DNA, all enzymatic
  reactions stop .
• Annealing at 54? The more stable bonds last a
  little bit longer (primers that fit exactly) and
  on that little piece of double stranded DNA
  (template and primer), the polymerase can attach
  and starts copying the template.
• Extension at 72? This is the ideal working
  temperature for the polymerase. The bases
  (complementary to the template) are coupled to
  the primer on the 3' side (the polymerase adds
  dNTP's from 5' to 3', reading the template from
  3' to 5' side, bases are added complementary to
  the template)

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NUCLEIC ACIDS
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How to determine the sequence of bases in a DNA
molecule
NUCLEIC ACIDS

• The most commonly used method of sequencing DNA -
  the dideoxy or chain termination method - was
  developed by Fred Sanger in 1977 (for which he
  won his second Nobel Prize). The key to the
  method is the use of modified bases called
  dideoxy bases when a piece of DNA is being
  replicated and a dideoxy base is incorporated
  into the new chain, it stops the replication
  reaction.

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The Nobel Prize in Chemistry 1980
NUCLEIC ACIDS
For more, click http//nobelprize.org
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Elements
NUCLEIC ACIDS

• The DNA to be sequenced in single-stranded form
  as a template.
• The four nucleotides
• The enzyme DNA polymerase and a primer
• A nucleotide analogue that cannot be extended and
  thus acts as a chain terminator

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NUCLEIC ACIDS
Dideoxynucleotides used in DNA sequencing
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NUCLEIC ACIDS
Train termination in the presence of
dideoxynucleotides
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Mechanism
NUCLEIC ACIDS
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NUCLEIC ACIDS
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One example of fluorecent chain-terminating
nucleotides
NUCLEIC ACIDS
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Sequencing Whole Genomes
NUCLEIC ACIDS
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NUCLEIC ACIDS

• First, the source clone is fragmented, producing
  a random mixture, and a random sub-clone is
  selected for sequencing by the Sanger method.
• To ensure that that the whole source clone has
  been sequenced, this stretch of DNA must be
  sequenced numerous times to produce an ordered
  overlapping sequence.
• Gaps in this process will occur where a sub-clone
  is not fully sequenced.

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Contigs
NUCLEIC ACIDS

• Assemble the short sequences from random shotgun
  DNAs into larger contiguous sequences.

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NUCLEIC ACIDS
Contigs are linked by sequencing the ends of
large DNA fragments
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Genome-wide analyses
NUCLEIC ACIDS

• Animal genomes contain complex exon-intron
  structure, so it is more difficult to find
  protein coding genes.

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NUCLEIC ACIDS

• A variety of bioinformatics tools are required to
  identify genes and determine the genetic
  composition of complex genomes.
• A notable limitation of current gene finder
  programs is the failure to identify promoters
• EST (expressed sequence tag) is simply a short
  sequence read from a larger cDNA.

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NUCLEIC ACIDS
Gene finder methods Analysis of proteincoding
regions in Ciona
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Comparative Genome Analysis
NUCLEIC ACIDS

• Permits a direct assessment of changes in gene
  structure and sequence arisen during evolution.
• Refines the identification of protein-coding
  genes within a given genome.

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What we have learned from comparative genome
analysis
NUCLEIC ACIDS

• Synteny conservation in genetic linkage, between
  distantly related animals.

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Part II PROTEINS
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Purification of proteins
PROTEINS

• To purify proteins we make use of their inherent
  similarities and differences.
• Protein similarity is used to purify them away
  from the other non-protein contaminants.
• Differences are used to purify one protein from
  another. Proteins vary from each other in size,
  shape, charge, hydrophobicity, solubility, and
  biological activity.

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ImmunoAffinity Chromatography
PROTEINS
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Affinity Chromatography
PROTEINS

• column matrix has a ligand that specifically
  binds a protein
• specialty affinity columns for binding
  recombinant proteins with certain "tags"

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Affinity Chromatography
PROTEINS
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Ion Exchange Chromatography
PROTEINS

• proteins have charges due to amino acid side
  groups
• bind to charged column matrix depending on their
  charge at a particular pH
• anionic--negatively charged phosphocellulose,
  heparin sepharose, S-sepharose
• cationic--positively charged DEAE-sepharose,
  Q-sepharose
• elute bound proteins from column based on charge
  and displacement by salt or pH

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Ion Exchange Chromatography
PROTEINS
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Gel filtrationChromatography
PROTEINS
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Separation of proteins on polyacrylamide gels
PROTEINS
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PROTEINS

• Proteins to be isolated should be treated with
  sodium dodecyl sulphate (SDS) and a reducing
  agent first to eliminate the secondary, tertiary,
  and quarternary structure.

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Protein molecules can be directly sequenced.
PROTEINS

• Edman degradation
• Tandem mass spectrometry

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Edman degradation
PROTEINS

• PITC is used to derivitize the free N-terminus
• trifluoroacetic acid causes cleavage of the
  N-terminal amino acid from the protein
• acid treatment rearranges derivitized aa to
  stable PTH amino acid
• the PTH amino acid is separated by chromatography
  (HPLC) and identified
• N-terminus may be subjected to another round of
  degradation

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PROTEINS
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Tandem mass spectrometry
PROTEINS
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Proteomics
PROTEINS

• Proteomics is the large-scale study of proteins,
  particularly their structures and functions. This
  term was coined to make an analogy with genomics.
  
• The availability of whole genome sequences in
  combination with analytic methods for protein
  separation and identification has ushered in the
  field of proteomics.

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Proteomics is based on three principal methods
PROTEINS

• 2-D gel electrophoresis for protein separation
• Mass spectrometry for the precise determination
  of the molecular weight and identity of a protein
• Bioinformatics for assigning proteins and
  peptides to the predicted products of protein
  coding sequences in the genome.

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