The Code For Life

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The Code For Life
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The Code For Life
Big nose
Brown eyes
Straight hair
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Structural Biology Medicine and Biology at the
Atomic Scale
Organ ? Tissue ? Cell ? Molecule ? Atoms

• A cell is an organization of millions of
  molecules
• Proper communication between these molecules is
  essential to the normal functioning of the cell
• To understand communication between molecules
  determine the arrangement of the atoms

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Advanced Cell Developmental Biology
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• Gene, Recombinant DNA Cloning Analysis

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Restriction Enzymes

• Restriction enzymes are DNases (nucleases) found
  in bacteria that recognize specific DNA sequences
  as 4mers,6mers or 8mers and make double stranded
  breaks in DNA .
• This enables cutting of genome in specific ways
  to generate restriction site maps and the
  development of approaches for pasting pieces of
  DNA together in specific ways.

A
Separation of EcoR1 segments on an agarose gel
B
C
D ,E
F
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DNA Hybridization

• DNA hybridization is the process whereby
  complementary strand of DNA anneals (to form a
  double helix) with the single stranded DNA
• Hybridization can be measured by labeling the
  complementary strand either with 32P
  nucleotides or fluorescent probes .
• There is also DNA-RNA hybridization

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Southern Blotting

• Southern Blotting enables identification of
  specific DNA sequences (gene
• fragments) from among the total sequence of DNA
  

Hybridize with a labeled DNA or RNA of interest (
e.g., 32P labeled DNA) followed by
autoradiography or phosphoimaging for detection
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Northern Blotting

• Northern Blotting is where RNA is blotted and
  then probed labeled DNA (cDNA)
• synthesized from the mRNA isolated from the
  cell
• Enables identification and quantification of
  specific mRNAs from among the vast
• population of RNAs in the cell

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DNA cloning

• DNA cloning enables specific pieces of genome to
  be inserted into bacteria as plasmid or phage
  lambda vectors and grown in large quantity.
• The first step is to generate a library of
  bacteria with inserted DNA fragments. This could
  either be a genomic(DNA)or a cDNA (mRNA) library

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Replica plating and in situ hybridization

• Techniques used to identify a bacterial colony
  that contains the gene (DNA sequence)
• of interest. The isolated colony can be grown
  up in large quantities.

CsCl centrifugation for separation of plamid DNA
from chromosomal DNA
Replica plating and in situ hybridization
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cDNA libraries

• They are generated to isolate particular genes
  of interest or to identify a gene based on the
  protein expression of that gene cloned in the
  bacterial cell
• The latter procedure is called reverse
  genetics whereby the protein product is used to
  identify the gene followed by DNA sequencing

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DNA sequencing

• Sangers dideoxy method DNA to be sequenced is
  mixed with each of 4 ddNTPS (chain terminators)
  in separate reactions for DNA synthesis and later
  separation of the products by electrophoresis
• Can now be done automatically via sequencing
  machines that work with different flurochromes
  attached to each of dideoxy nucleotides
• To determine the sequence of a gene of many
  kilobases overlapping DNA fragments of 400-800 bp
  must be sequenced

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Protein expression vectors

• These are specially designed plasmid
• vectors for fusion protein expression
• to isolate large quantities of protein of
• interest for antibody production or
• other studies of purified protein.
• The proteins are produced as fusion
• proteins of the cDNA gene coding
• sequence ligated to a protein
• expression marker or reporter protein
• e.g. beta-galactosidase
• They can also be used as a major tool
• in cell biology to study the expression
• of proteins in cells following DNA
• transfection

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DNA transfection and Polymerase chain reaction
(PCR)

• DNA transfection is used to track the properties
  of individual proteins in a cell
• Construct a plasmid expression system that
  contains the protein of interest fused with a
  reporter gene such as a beta- galactosidase or a
  short peptide sequence such as HA 9 mer peptide
  or FLAG epitope for antibody localization with
  anti HA or anti FLAG or fluorescent localization
  in living cells with GFP-constructs (GFP-actin)

Polymerase chain reaction (PCR) Is used as an
alternative to cloning for purifying a particular
DNA (gene sequence It enables the production of
microgram quantities of the DNA sequence of
interest in the test tube Provides an
alternative for preparing DNA probes to screen
genomic or cDNA library for clones encoding a
protein of interest
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DNA Microarrays and chips

• Enable via fluorescence in situ hybridization
  (FISH) to measure expression of 1000s of genes
  on each array/ chip.

Actual chip size
Yeast genome microarray The array is hybridized
to cDNA labeled with a green fluorescent dye
prepared from cells grown in glucose and with red
labeled cDNA from cells grown in ethanol. Spots
were detected with a scanning confocal microscope

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Antibody production

• Polyclonal antibodies are
• generated by injecting
• antigen into an animal and
• purifying the antibody
• titer from blood
• Monoclonal antibody
• technique enables to obtain
• a single clone of cells that
• recognizes one epitope
• ( usually 9 a.a.) of the
• total protein

Monoclonal antibody production
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Genetic Engineering

• Introduction of exogenous genes ( mutant or
  normal) in to normal cells or organisms to study
  gene expression
• Used to study the role of the protein coded by
  the gene in the cell/organism function or for
  engineering gene expression for improving food
  production or reducing the destrcutive damage of
  human diseases

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Site Directed Mutagenesis

• Alterations in nucleotides (substitutions or
  deletions) in vitro at known (directed) sites to
  create mutant genes
• These mutant genes can be transfected into cells
  as previously discussed and enables study of gene
  function at the individual cell level. The
  transfected genes are also called transgenes

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Production of transgenic mouse
Inject mutant gene in to one of the pronuclei of
the fertilized mouse oocyte
Transfer oocyte to surrogate mother. 10-30 of
offspring contain the transgene in equal amounts
in all tissues
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Gene Knockout or replacement

• Form of trangenics
• Occurs following homologous recombination of the
  transgene at the site of the endogenous gene
• Occurs readily in yeast cells but in mammalian
  cells the rate of recombination is very slow and
  hence a double selection marker approach is
  adopted where the first marker e.g. neomycin
  resistance selects for all cells with homologous
  recombination while the second marker allows
  growth of only those cells that carried out
  homologous recombination

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Knockout protocol
ES cells are isolated from the inner blastocyst
and culture
ES cells are tranfected with the gene of interest
Enables direct study of gene function in an
intact organism
ES cells successfully transfected via homologous
recombination are selected and grown in culture
and injected into a host blastocyst. Chimeras
develop which contain ES cells from both the
transfected and the host cells.
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Gene Replacement/therapy

• Replace an abnormal
• gene with a normal one
• at a very early stage of
• development
• It has the potential for
• curing or alleviating the
• symptoms of a wide
• variety of human
• diseases, e.g.,Parkinsons
• disease

Procedure for gene replacement
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How Ian Wilmut Made Dolly 1Making Quiescent Cells
Culture mammary cells
Starve cells
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How Ian Wilmut Made Dolly 2Collecting The Donor
Nucleus
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How Ian Wilmut Made Dolly 2Collecting The Donor
Nucleus
Glass pipette
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How Ian Wilmut Made Dolly 3Egg Preparation
An egg is collected then placed into a dish where
it can be manipulated
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How Ian Wilmut Made Dolly 3Egg Preparation
Egg
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How Ian Wilmut Made Dolly 3Egg Preparation
Glass pipette
Egg
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How Ian Wilmut Made Dolly 4Inserting The Donor
Nucleus
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How Ian Wilmut Made Dolly 4Inserting The Donor
Nucleus
Glass pipette
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How Ian Wilmut Made Dolly 4Inserting The Donor
Nucleus
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How Ian Wilmut Made Dolly 5Initiating Development
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How Ian Wilmut Made Dolly 5Initiating Development
Zygote
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How Ian Wilmut Made Dolly 5Initiating Development
Cleavage
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How Ian Wilmut Made Dolly 5Initiating Development
Cleavage
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How Ian Wilmut Made Dolly 5Initiating Development
Cleavage
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How Ian Wilmut Made Dolly 5Initiating Development
Cleavage
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How Ian Wilmut Made Dolly 5Initiating Development
Morula
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How Ian Wilmut Made Dolly 6Development