Genetic at glance

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King Fahd University of Petroleum Mineral
Graduate Seminar
Genetic at glance
Al-Khadem Yousef Ahmad December 30,
2007
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Outline

• The chromosome as control tool .

• The strategies of DNA manipulation

• Utilizations of genetic engineering in Medicine,
  agriculture

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• Gene and protein biosynthesis

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Nucleotide The phosphate groups (PO43-) and the
pentose sugars form the backbone of both DNA and
RNA.
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The purine base and pyrimidine of DNA
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Hydrogen Bond (H-Bond)
The linearity is important for a perfect H-bond

Weaker H-bond
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The Central Dogma
From Gene to Protein Prokaryotes
Transcription Translation Eukaryotes
Transcription mRNA Modification Translation
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TRANSCRIPTION RNA-polymerase-catalyzed synthesis
of RNA from a DNA template.
Transcription Proceeds in three distinct
phases 1. Initiation (binding of RNA polymerase
to template DNA). 2. Elongation (nucleotides are
added to the DNA template). 3. Termination (the
enzyme and RNA is release from DNA template)
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Transcription produces pre-mRNA that
contains both introns exons
5 cap poly-A tail are added to pre-mRNA RNA
splicing removes intones mRNA is made
Initiation occurs in a specific region on the
DNA called the promoter.
The promoter contains sequence elements that bind
several transcription factors in combination with
RNA polymerase
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Elongation

• elongation begins and RNA nucleotides add
  sequentially until

• the polymerase reaches the end of the template

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Termination
Sequences signaling termination
A Simple Gene
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RNA PROCESSING PRE-mRNA ?? mRNA
1) Synthesis of the cap modified G which is
attached to the 5 end of the pre-mRNA
2) Removal of introns
3) Synthesis of the poly(A) tail transcript is
cut at a site when completed, and a poly(A) tail
is attached to the exposed 3 end.
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TRANSLATION

• The ribosome binds to the mRNA at the start
• cordon (AUG) that is recognized only by the
• initiator tRNA.

• ?? Complexes, composed of an amino acid
• linked to tRNA, sequentially bind to the
• appropriate codon in mRNA by forming
• complementary base pairs with the tRNA
• anticodon.

• ?? Amino acids are added one by one,
• translated into polypeptide sequences dictated
• by DNA and represented by mRNA.

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L-Form Amino Acid Structure
Carboxylic group
Amino group
H Glycine
R group
CH3 Alanine
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Peptide bonds

• Carboxyl group - linked to amino group of another
  amino acid.
• Many amino acids joined together is a
  polypeptide.
• Sequence read from Amino to carboxyl end
• Variable part side chains

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• The trans conformation of the peptide bond is
  favored over the cis conformer due to steric
  hinderance between side chains

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How to deal with genetic concept in real

• Technique involving the insertion of a fragment
  of foreign DNA into a vector capable of
  replicating autonomously in a host cell (usually
  Escherichia coli).

• Growing the host cell allows the production of
  multiple copies of the inserted DNA for use in a
  variety of purposes.

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Requirements for Molecular Cloning

• Vector DNA for cloning

• Means of inserting foreign DNA into the vector

• Method Foreign DNA

• of placing the in vitro modified DNA into the
  host cell

• Methods for selecting and/or screening cells
  that carry the inserted foreign DNA

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Enzymes used in molecular cloning
Restriction Endonucleases
Type I Long recognition sequences and random
to cleavage Type II Tetra, Penta and Hexa
recognition sequences Type III Similar to type
I but specific to cleavages
Polymerases
DNA Polymerase catalyzes the polymerization of
deoxyribonucleotides along the template
strand DNA-dependent RNA Polymerase
Nucleases
Enzymes capable of cleaving the phosphodiester
bonds between nucleotide subunits of nucleic acids
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Other Modifying Enzymes

• Ligases

forms phosphodiester bonds to join two pieces of
DNA utilizes ATP in the presence of Mg

• Kinases

• Phosphatases

catalyzes the removal of 5-phosphate residues
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Sources of Foreign DNA
Foreign DNA

• Genomic DNA

• Complementary DNA (cDNA)

• Synthetic DNA
• Chemical Synthesis
• Polymerase Chain Reaction (PCR)

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Cloning vectors

• Vector DNA

• DNA molecule that functions as a molecular
  carrier that carry the DNA of interest into the
  host cell facilitates its replication.

• Plasmids

• Bacteriophage

• Cosmids

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Inserting the DNA into the vector

• Means of inserting foreign DNA into the vector

Ligation of the DNA into the linearized vector
Requirements for a ligation reaction

• two fragments of DNA (blunt/cohesive)
• buffer containing ATP
• T4 DNA ligase

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Short gun cloning step
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Thank you