Introduction To Molecular Biology

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Introduction To Molecular Biology By Salwa Hassan
Teama (M.D)

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Molecular Biology

• Molecular biology the study of biology at the
  molecular level.
• Molecular biology the study of gene structure
  and functions at the molecular level to
  understand the molecular basis of hereditary,
  genetic variation, and the expression patterns of
  genes.
• The Molecular biology field overlaps with other
  areas, particularly genetics and biochemistry.

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The Genome

• The genome of an organism is the totality of
  genetic information and is encoded in the DNA
  (or, for some viruses, RNA).

commons.wikimedia.org/wiki/ImageGenome.jpg
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Genome Database

• The database is organized in six major organism
  groups
• Eukaryotes, Bacteria, Archaea, Viruses, Viroids
  and Plasmids.

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Three Domain of Life

• All living things are grouped into three domain
• Eukaryotes
• Prokaryotes and
• Archaea.

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The Cell

• The cell is the smallest living unit, the
  basic structural and functional unit of all
  living things. Some organisms, such as most
  bacteria, are unicellular (consist of a single
  cell). Other organisms, such as humans, are
  multicellular.

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The Cell

• Cells are stacked together to make up structures,
  tissues and organs. Most cells have got the same
  information and resources and the same basic
  material. Cells can take many shapes depending on
  their function.

• Function of cells
• Secretion (Produce enzymes).
• Store sugars or fat.
• Brain cells for memory and intelligence.
• Muscle cells to contract.
• Skin cell to perform a protective coating.
• Defense, such as white blood cells.

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Eukaryotic Cell

• Eukaryotes are generally more advanced than
  prokaryotes. There are many unicellular organisms
  which are eukaryotic, but all cells in
  multicellular organisms are eukaryotic.
• Eukaryotic cells are found in animals plants
  fungi and protists cell.

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Eukaryotic Cell

• Cell with a true nucleus, where the genetic
  material is surrounded by a membrane
• Eukaryotic genome is more complex than that of
  prokaryotes and distributed among multiple
  chromosomes
• Eukaryotic DNA is linear
• Eukaryotic DNA is complexed with proteins called
  "histones
• Numerous membrane-bound organelles
• Complex internal structure
• Cell division by mitosis.

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Prokaryotic Cell

• Unicellular organisms, found in all environments.
  These include bacteria and archaea.
• Without a nucleus no nuclear membrane (genetic
  material dispersed throughout cytoplasm
• No membrane-bound organelles
• Cell contains only one circular DNA molecule
  contained in the cytoplasm
• DNA is naked (no histone)
• Simple internal structure and
• Cell division by simple binary fission.

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Archaea

• Archaea are prokaryotes organisms without
  nucleus but some aspect of their molecular
  biology are more similar to those of eukaryotes.

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Eukaryotic Cell Cycle

• Eukaryotic Cell Cycle defined as the
  sequence of events that occurs during the
  lifetime of a cell and is traditionally divided
  into four phases
• G1 Growth and preparation of the chromosomes
  for replication
• S Synthesis of DNA
• G2 Preparation for mitosis
• M Mitosis

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Central Dogma of Molecular Biology

• The flow of genetic information as follows

http//www.emc.maricopa.edu/faculty/farabee/BIOBK/
BioBookPROTSYn.html
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Deoxyribonucleic Acid (DNA)

• Deoxyribonucleic Acid (DNA), the genetic
  material of all cellular organisms and most
  viruses, the gigantic molecule which is used to
  encode genetic information for all life on Earth.

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Eukaryotic Cell
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http//genome.gsc.riken.go.jp/hgmis/graphics/slide
s/01-0085jpg.html U.S. Department of Energy
Human Genome Program, http//www.ornl.gov/hgmis.
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The Chromosome

• Thread like structure.
• Located in the cell nucleus.
• The storage place for all genetic information.
• The number of chromosomes varies from one
  species to another.

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The Chromosome

• In normal human cell DNA contained in the
  nucleus, arranged in 23 pairs of chromosomes 22
  pairs of chromosomes (autosomes) the 23
  chromosome pair determines the sex of individual
  and is composed of either two (x) chromosomes
  (female) or an (x) and (y) chromosome (male).

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The Gene

• The basic units of inheritance it is a segment
  within a very long strand of DNA with specific
  instruction for the production of one specific
  protein. Genes located on chromosome on it's
  place or locus.

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General Structure of Nucleic Acid

• DNA and RNA are long chain polymers of small
  compound called nucleotides. Each nucleotide is
  composed of a base sugar (ribose in RNA or
  deoxyribose in DNA) and a phosphate group. The
  phosphate joins the sugars in a DNA or RNA chain
  through their 5 and 3 hydroxyl group by
  phosphodiester bonds.

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• The structure of DNA was described by British
  Scientists Watson and Crick as long double helix
  shaped with its sugar phosphate backbone on the
  outside and its bases on inside the two strand
  of helix run in opposite direction and are
  anti-parallel to each other. The DNA double helix
  is stabilized by hydrogen bonds between the
  bases.
• This structure explains how genes engage in
  replication, carrying information and acquiring
  mutation.
• The GC content of a natural DNA can vary from
  22-73 and this can have a strong effect on the
  physical properties of DNA, particularly its
  melting temperature.

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• There are four different types of nucleotides
  found in DNA, differing only in the nitrogenous
  base A is for adenine G is for guanine C is
  for cytosine and T is for thymine.
• These bases are classified based on their
  chemical structures into two groups adenine and
  guanine are double ringed structure termed purine
  , thymine and cytosine are single ring structures
  termed pyrimidine.
• The bases pair in a specific way Adenine A with
  thymine T (two hydrogen bonds) and guanine G
  with cytosine C (three hydrogen bonds).
• Within the structure of DNA, the number of
  thymine is always equal to the number of adenine
  and the number of cytosine is always equal to
  guanine.
• In contrast to DNA RNA is a single stranded,
  the pyrimidine base uracil (U) replaces thymine
  and ribose sugar replaces deoxyribose.

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Genomic DNA organization

• Eukaryotic genes DNA molecules complexed
  with other proteins especially basic proteins
  called histones, to form a substance known as
  chromatin. A human cell contains about 2 meters
  of DNA. DNA in body could stretch to the sun and
  back almost 100 times. So it is tightly packed.

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Eukaryotic Chromatin

• Eukaryotic chromatin is folded in several ways.
  The first order of folding involves structures
  called nucleosomes, which have a core of
  histones, around which the DNA winds ( four pairs
  of histones H2A, H2B,H3 and H4 in a wedge shaped
  disc, around it wrapped a stretch of 147 bp of
  DNA).

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DNA Forms


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DNA Replication

• DNA Replication The DNA (all gene) duplication
  the transfer the genetic information from a
  parent to a daughter cell the DNA base sequence
  are precisely copied.
• Replication proceeds in a semiconservative
  manner, each strand of the DNA helix serves as a
  template for the synthesis of complementary DNA
  strands. This lead to the formation of two
  complete copies of the DNA molecule, each
  consisting of one strand derived from the parent
  DNA molecule and one newly synthesized
  complementary strand.

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Mitochondrial DNA

• Mitochondria is a membrane-enclosed organelle
  found in most eukaryotic cells.These organelles
  range from 110 micrometers (µm) in size.
• Mitochondria generate most of the cell's supply
  of adenosine triphosphate (ATP).
• Mitochondria are involved in a range of other
  processes, such as signaling, cellular
  differentiation, cell death, as well as the
  control of the cell cycle and cell growth.
• Mitochondria have been implicated in several
  human diseases, including mental
  disorders,cardiac dysfunction,and may play a
  role in the aging process.
• Mitochondria has its own DNA.

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Mitochondrial DNA

• Mitochondrial DNA contains 37 genes, all of which
  are essential for normal mitochondrial function.
  Thirteen of these genes provide instructions for
  making enzymes involved in oxidative
  phosphorylation.
• Oxidative phosphorylation is a process that uses
  oxygen and simple sugars to create adenosine
  triphosphate (ATP), the cell's main energy
  source.
• The remaining genes provide instructions for
  making molecules called transfer RNAs (tRNAs) and
  ribosomal RNAs (rRNAs).
• Mitochondrial genes are among the estimated
  20,000 to 25,000 total genes in the human genome.

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Function of The DNA

• Deoxyribonucleic Acid (DNA), the gigantic
  molecule which is used to encode genetic
  information for all life on Earth.
• The chemical basis of hereditary and genetic
  variation are related to DNA.
• DNA directs the synthesis of RNA which in turn
  directs protein synthesis.

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The Genetic Code

• The purine and pyrmidine bases of the DNA
  molecule are the letters or alphabet of the
  genetic code. All information contained in DNA
  represented by four letters A,T,C,G.
• Three nucleotides of DNA (1st, 2nd and 3rd) form
  triplet codons. A group of codons constitute the
  genetic code, that can be translated into amino
  acid of proteins.
• RNA Codon tRNA Amino Acids

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The Genetic Code

• The sequence of codons in the mRNA defines the
  primary structure of the final protein. Since
  there are 64 possible codons, most amino acids
  have more than one possible codon. Out of the 64
  possible 3-base codons, 61 specify amino acids
  the other three are stop signals (UAG, UAA, or
  UGA).

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The RNA

• Three major classes of RNA messenger (mRNA),
  transfer (tRNA) and ribosomal (rRNA). Minor
  classes of RNA include small nuclear RNA small
  nucleolar RNA..

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The RNA

• The concentration of purine and pyrimidine bases
  do not necessarily equal one another in RNA
  because RNA is single stranded. However, the
  single strand of RNA is capable of folding back
  on itself like a hairpin and acquiring double
  strand structure.

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Messenger RNA

• mRNA molecules represent transcripts of
  structural genes that encode all the information
  necessary for the synthesis of a single type
  polypeptide of protein.
• mRNA intermediate carrier of genetic
  information deliver genetic information to the
  cytoplasm where protein synthesis take place.
• The mRNA also contains regions that are not
  translated in eukaryotes this includes the 5'
  untranslated region, 3' untranslated region, 5'
  capand poly-A tail.

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Transfer RNA(tRNA)

• All tRNAs share a common secondary structure
  represented by a coverleaf. They have four-paired
  stems defining three stem loops (the D loop,
  anticodon loop, and T loop) and the acceptor stem
  to which amino acids are added in the charging
  step.
• RNA molecules that carry amino acids to the
  growing polypeptide.

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Ribosomal RNA (rRNA)

• Ribosomal RNA (rRNA) is the central component
  of the ribosome, the function of the rRNA is to
  provide a mechanism for decoding mRNA into amino
  acids and to interact with the tRNAs during
  translation by providing peptidyl transferase
  activity.

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Ribosomes

• Ribosomes Factory for protein synthesis are
  composed of ribosomal RNA and ribosomal proteins
  (known as a Ribonucleoproteinor RNP). They
  translate messenger RNA (mRNA) to build
  polypeptide chains using amino acids delivered by
  transfer RNA (tRNA).

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Ribosomes

• Eukaryotic ribosomes are larger. They consist of
  two subunits a 60S subunit holds (three rRNAs
  5S, 5.8S, 28S and about 40 proteins) and a 40S
  subunit contains (an18S rRNA and about 30
  proteins) , which come together to form an 80S
  particle compared with prokaryotic 70S ribosome

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Polysomes

• Most mRNA are translated by more than one
  ribosome at a time the result, a structure in
  which many ribosomes translate an mRNA in tandem,
  is called a polysomes.

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The Protein

• Proteins are the basic building materials of a
  cell, made by cell itself the final product of
  most genes.
• Proteins are chain like polymers of a few or many
  thousands of amino acids. Amino acids are
  represented by codons, which are 3-nucleotide RNA
  sequences. Amino acids joined together by peptide
  bonds (polypeptide). Proteins can be composed of
  one or more polypeptide chains.
• Proteins have many functions provide structure
  that help cells integrity and shape (e.g.
  collagen in bone) serve as enzymes and hormones
  bind and carry substance and control of
  activities of genes.

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Four levels of a protein's structure

• Primary structure Formed by joining the amino
  acid sequence into a polypeptide.
• Secondary structure Different conformation that
  can be taken by the polypeptide alpha helix and
  strands of beta sheet.
• Tertiary structure Result from folding the
  secondary structure components of the polypeptide
  into three-dimensional configuration.
• Quaternary structure complex of several protein
  molecules or polypeptide chains, usually called
  protein subunits, which function as part of the
  larger assembly or protein complex.

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Protein Structure
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Gene Expression

• Gene expression process by which a gene product
  (an RNA or polypeptide ) is made.
• In transcription steps, RNA polymerase make a
  copy of information in the gene (complementary
  RNA) (mRNA) complementary to one strands of DNA.
  
• In translation step, ribosomes read a messenger
  RNA and make protein according to its
  instruction. Thus any change in gene sequence may
  lead to change in the protein product.

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Types of control in Eukaryotes

• Transcriptional, prevent transcription, prevent
  mRNA from being synthesized.
• Posttranscriptional, control mRNA after it has
  been produced.
• Translational, prevent translation involve
  protein factors needed for translation.
• Posttranslational, after the protein has been
  produced.

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Mutation

• Mutation include both gross alteration of
  chromosome and more subtle alteration to specific
  gene sequence.
• Gross chromosomal aberrations include large
  deletions addition and translocation (reciprocal
  and nonreciprocal).
• Mutation in a gene's DNA sequence can alter the
  amino acid sequence of the protein encoded by the
  gene. Point mutations are the result of the
  substitution of a single base. Frame-shift
  mutations occur when the reading frame of the
  gene is shifted by addition or deletion of one or
  more bases.

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Mutation

• Mutations can have harmful, beneficial,
  neutral, or uncertain effects on health and may
  be inherited as autosomal dominant, autosomal
  recessive, or X-linked traits. Mutations that
  cause serious disability early in life are
  usually rare because of their adverse effect on
  life expectancy and reproduction.

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Common Tools in Molecular Biology

• Nucleic acid fractionation
• Polymerase chain reaction
• Probes, Hybridization
• Vector, Molecular cloning
• Nucleic acid enzymes
• Microarray
• DNA sequencing
• Electrophoretic separation of nucleic acid
• Detection of genes
• DNA Southern blotting inSitu
  hybridization FISH Technique
• RNA Northern blotting
• Protein Western blotting,
  immunohistochemistry

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Human Genome Project

• Goals
• Identify all the approximately 20,000-25,000
  genes in human DNA,
• Determine the sequences of the 3 billion chemical
  base pairs that make up human DNA, store this
  information in databases,
• Improve tools for data analysis, transfer
  related technologies to the private sector, and
• Address the ethical, legal, and social issues
  (ELSI) that may arise from the project.

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Molecular Biology Uses

• Various methods in molecular biology diagnose the
  different human diseases diagnosis of an
  infectious agent, in malignancy, the presence of
  the genetic disease and in transplantation,
  paternity and forensic analysis.
• The Most Recent Applied Technologies
• Genetic engineering
• DNA finger-printing in the social and forensic
  science.
• Pre and postnatal diagnosis of inherited
  diseases.
• Gene therapy.
• Drug Design.

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Molecular biology is facilitating research in
many field including biochemistry, microbiology,
immunology and genetics, Molecular
biology allows the laboratory to be predictive in
nature, it gives information that the patients
may be at risk for disease (future).
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Glossary

• Alleles are forms of the same gene with small
  differences in their sequence of DNA bases.
• Exon (Coding DNA) A gene sequence contains
  protein coding information.
• Introns (intervening sequence) (A noncoding DNA
  sequence ) Intervening stretches of DNA that
  separate exons.
• Primary transcript The initial production of
  gene transcription in the nucleus an RNA
  containing copies of all exons and introns.
• RNA gene or non-coding RNA gene RNA molecule
  that is not translated into a protein. Noncoding
  RNA genes produce transcripts that exert their
  function without ever producing proteins.
  Non-coding RNA genes include transfer RNA (tRNA)
  and ribosomal RNA (rRNA), small RNAs such as
  snoRNAs, microRNAs, siRNAsand piRNAs and lastly
  long ncRNAs.
• Enhancers and silencers are DNA elements that
  stimulate or depress the transcription of
  associated genes they rely on tissue specific
  binding proteins for their activities sometimes
  a DNA elements can act either as an enhancer or
  silencer depending on what is bound to it.
• Activators Additional gene-specific
  transcription factors that can bind to enhancer
  and help in transcription activation.
• Open reading frame (ORF) A reading frame that
  is uninterrupted by translation stop codon
  (reading frame that contains a start codonand the
  subsequent translated region, but no stop codon).
  
• Directionality in molecular biology, refers to
  the end-to-end chemical orientation of a single
  strand of nucleic acid. The chemical convention
  of naming carbon atoms in the nucleotide
  sugar-ring numerically gives rise to a 5' end and
  a 3' end ( "five prime end" and "three prime
  end"). The relative positions of structures along
  a strand of nucleic acid, including genes,
  transcription factors, and polymerases are
  usually noted as being either upstream (towards
  the 5' end) or downstream (towards the 3' end).
• 3' flanking region Present adjacent to 3' end of
  the gene often contain sequences which affect
  the formation of the 3 end of the message and
  may contain enhancers or protein binding sites.
• 5' flanking region A region adjacent to 5' end
  of the gene. It is not transcribed into RNA it
  contains the promoter. May contain enhancers or
  other protein binding sites.
• 3' untranslated region The three prime
  untranslated region (3' UTR) is a particular
  section of messenger RNA (mRNA). It follows the
  coding region. It is a region of the DNA which is
  transcribed into mRNA and becomes the 3' end or
  the message, Several regulatory sequences are
  found in the 3' UTR. The 3' untranslated region
  may affect the translation efficiency of the mRNA
  or the stability of the mRNA. It also has
  sequences which are required for the addition of
  the poly(A) tail to the message (including one
  known as the "hexanucleotide", AAUAAA).
• 5' untranslated region The five prime
  untranslated region (5' UTR), also known as the
  leader sequence, is a particular section of
  messenger RNA (mRNA) and the DNA that codes for
  it. It is a region of a gene which is transcribed
  into mRNA. It starts at the site (where
  transcription begins) and ends just before the
  start codon (usually AUG) of the coding region.
  It usually contains a ribosome binding site
  (RBS), in bacteria also known as the Shine
  Dalgarno sequence (AGGAGGU). In prokaryotic mRNA
  the 5' UTR is normally short. Some viruses and
  cellular genes have unusual long structured 5'
  UTRs which may have roles in gene expression.
  Several regulatory sequences may be found in the
  5' UTR.
• Reverse Transcription Some viruses (such as HIV,
  the cause of AIDS), have the ability to
  transcribe RNA into DNA.

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References Online Further Reading

• Robert F. Weaver. Molecular Biology. Fourth
  Edition. Page 600. McGraw-Hill International
  Edition. ISBN 978-0-07-110216-2
• Innis,David H. Gelfand,John J. Sninsky PCR
  Applications Protocols for Functional Genomics
  ISBN0123721865
• Daniel H. Farkas. DNA Simplified The
  Hitchhiker's Guide to DNA. Washington, DC AACC
  Press, 1996, ISBN 0-915274-84-1.
• William B. Coleman,Gregory J. Tsongalis
  Molecular Diagnostics For the Clinical
  Laboratorian ISBN 1588293564...
• Robert F. Mueller,Ian D. Young. Emery's
  Elements of Medical Genetics ISBN. 044307125X
• Daniel P. Stites,Abba T. Terr. Basic Human
  Immunology ISBN. 0838505430
• Bruce Alberts, Alexander Johnson, Julian Lewis,
  Martin Raff, Keith Roberts, and Peter Walter.
  Molecular Biology of the cell. ISBN.
  9780815341055
• http//www.pubmedcentral.nih.gov/
• http//www.biomedcentral.com/1471-2105/2/8/abstrac
  t. Elena Rivas and Sean R Eddy Noncoding RNA
  gene detection using comparative sequence
  analysis
• BMC Bioinformatics 2001,
  28doi10.1186/1471-2105-2-8
• www.medscape.com
• http//www.medterms.com/script/main/art.asp?articl
  ekey4026
• www.emedicine.com
• www.ebi.ac.uk/2can good introduction to
  bioinformatics and molecular biology
• http//www.genomicglossaries.com/
• http//www.gene.ucl.ac.uk/nomenclature/guidelines.
  html defines the nomenclature for human genes
• http//www.accessexcellence.org
• http//users.rcn.com/jkimball.ma.ultranet/BiologyP
  ages/C/Codons.html
• http//www.web-books.com/MoBio/

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Thank You