MOLECULAR BASIS OF INHERITANCE

Ø THE DNA (DEOXY – RIBO– NUCLEIC ACID):-

1.      Long polymer of De-oxy ribo nucleotides.

2.   Length of DNA is defined as the number of nucleotides {pentose/ deoxyribose sugar+ Phosphate bond + Nitrogenous base) or a pair of nucleotide (known as BASE PAIR or bp) present in it. [Characteristic of an organism/species].

3.  Bacteriophage φ x 174 has 5386 nucleotides; Lambda phage (λ-phage) has 48502 bp (double nucleotides).

4.  Escherichia coli has 4.6 x 10⁶ bp & Haploid (n) set of Human DNA is 3.3 x 10⁹ bp.

Ø STRUCTURE OF POLYNUCLEOTIDE CHAIN (DNA /RNA):-

       NUCLEOTIDE has a NITROGENOUS BASE [PURINE {Adenine/Guanine} & PYRIMIDINES {Cytosine/Thymine / Uracil}].  While Cytosine is common in both, Thymine is present ONLY in DNA (replaced by Uracil in RNA).

        Nitrogenous base is linked to a Pentose Sugar through a N – glycosidic linkage {NUCLEOSIDE} eg. Adenosine.

       The 5’-OH group of a Nucleoside linked to a phosphate group through Phosphodiester linkage {NUCLEOTIDE}.

(Sugar Phosphate Linkage)

       Two Nucleotides linked to a 3’-5’ phosphodiester linkage to form a DINUCLEOTIDE. More nucleotides may join to form polynucleotide, with a free Phosphate moiety at 5’-end of ribose sugar & 3’-OH group at 3’-end of sugar.

    RNA:-Sugar is Ribose (extra OH- group at C2 position of pentose ring). Thymine (5-Methyl Uracil)is replaced by URACIL. Single Stranded Nucleic acid, Highly Reactive, Polymorphic (mRNA, tRNA, and rRNA).

    DNA (as an acidic substance called nuclein –Fredrick Mistier, 1869).Watson – Crick (1953) proposed DOUBLE HELIX (based on X- ray diffraction studies of Wilkins & Franklin)

    SALIENT FEATURES OF DOUBLE – HELIX STRUCTURE OF DNA:-

1.     Two Anti parallel polynucleotide chains ( 5’ →3’ and 3’→5’).

2.     Chains with Backbone of Sugar Phosphate & Nitrogenous bases projecting inside.

3.     Base Pairing (bp) through Hydrogen Bonding (A=T and C ≡ G).  [Purine bonds with Pyrimidins]. This generated approx. equal bond length & symmetry to the molecular structure, hence stability).

4.     Two chains are coiled in a RIGHT HANDED FASHION (alpha helix) also known as B-DNA.  The Pitch of the helix is 34 A^o (A^o = 1 x 10 ^-10 m)  with 20bp in each turn. (Distance of 3.4 A^o   between two bp).

5.     The plane of one bp stacks over another, in a double helix, which provides additional stability.

6.     CHARGAFF’S RULE:-  (a) A=T and C ≡ G  (b) A:G = T:C)


             [ VIDEO ON WATSON - CRICK STRUCTURE OF DNA ]

    CENTRAL DOGMA (F. Crick) :  Genetic information flow as    DNA → RNA → PROTEIN.

    In some Viruses (with RNA as genetic material) REVERSE TRANSCRIPTION (TEMINISM) occurs.

    PACKAGING OF DNA HELIX (Nucleosome Concept)

                Length of DNA in a Typical Mammalian cell = Number of bp X Distance between two bp                                                     .                                                                                   = 6.6 x 10⁹ bp X 0.34x10^-9 m = ~2.2m.

                If length of E.coli is 1.36m, then number of bp = 1.36/0.34x10^-9 m = 4000000000 bp = 4 X 10⁹ bp.

NUCLEOSOME CONCEPT:

       In Eukaryotes, Histone Protein ( + Charged, basic protein, rich in basic amino acids Lysine & Arginine) organize as 8 molecules (HISTONE OCTAMER) – 2x( H2A, H2B,H3 & H4) + H1 bound from outside. Negatively charged DNA is wrapped around the core (containing 200 bp of DNA).

       NUCLEOSOME forms the repeating unit in nucleus called CHROMATIN (thread like stained body of DNA). It appears as a “string of beads”.

                                   [ VIDEO ON NUCLEOSOME CONCEPT ]

       How many such beads are present in a typical mammalian cell?                    {2n= 46; hence 46 beads}.

       Chromatin is packed as chromatin fibres further coiling (super-/ Hyper- coiling) at metaphase stage to appear as chromosome. Higher level packing requires Non Histone Chromosomal (NHC) Protein.

                                         

    EUCHROMATIN & HETEROCHROMATIN:-

Some regions of chromatin are loosely packed, transcriptionally Active & light stained (EUCHROMATIN). Chromatin that is densely packed, transcriptionally Inactive & stain dark (HETEROCHROMATIN)

                                                                [VIDEO ON EUCHROMATIN & HETEROCHROMATIN ]

    TRANSFORMING PRINCIPLE 

[ Fredrick Griffith, 1928]

1.     Experimented on Streptococcus pneumonia (avirulent R- Strain & virulent S- Strain).

2.     S-strain has a mucous/polysaccharide coat on it, hence when injected in mice, develop the disease.

3.     (EXPERIMENT-1) R-strain → injected in mice →Mice LIVED.

4.     (EXPERIMENT-2) S-strain → injected in mice →Mice DIED.

5.     (EXPERIMENT-3) S-strain (Heat Killed) → injected in mice →Mice LIVED.

6.     (EXPERIMENT-4) {R-strain  + S-strain (Heat Killed)} → injected in mice →Mice DIED. (live S bacteria recovered from the body of dead mice.

7.     CONCLUSION:- Griffith concluded that some Genetic Material transformed & enabled R-strain to synthesize a smooth polysaccharide coat & become Virulent.

                                      [ VIDEO ON GRIFFITH'S TRANSFORMATION EXPERIMENT ]

8.     BIOCHEMICAL CHARACTER OF TRANSFORMING PRINCIPLE: -

(Avery, MacLeod & McCarty, 1944) worked on ‘Griffith’s Principle of Transformation’. They purified biochemicals (Protein, DNA, RNA etc..) from heat killed S-bacterial cells & mixed with R-cells. Only DNA caused TRANSFORMATION. Also protein digesting enzymes & RNAase did not affect transformation. CONCLUSION: - Transforming material is DNA. Hence proved that DNA is the genetic material.

                                             [ VIDEO OF AVERY -McLEOID & McKARTY EXPERIMENT ]

                     GENETIC MATERIAL  IS  DNA
           (FINAL CONCLUSION)

                     (Martha Chase - Alfred Harshey, 1952)

1.     Hershey & Chase (1952) worked on Bacteriophages (Viruses infecting Bacteria)

2.     They grew viruses on medium containing Radioactive Sulphur (³⁵S) developing radioactive protein coat.

3.     They grew viruses on medium containing Radioactive Phosphorus (³²P) developing radioactive DNA.

4.     Radioactive Phages were allowed to attack E.coli bacteria & infection proceeds. Then, centrifuged in a blender to remove viral coat.

5.     Bacteria infected with radioactive phosphorus developed radioactive DNA & that with radioactive sulphur had developed no radioactivity (indicating that proteins did not entered bacteria from the virus).

6.     CONCLUSION:- DNA is the genetic material (that is passed from virus to bacteria).

                                                           [VIDEO OF HERSHEY-CHASE EXPERIMENT ]

    PROPERTIES OF GENETIC MATERIAL: (DNA vs. RNA)

1.     Should be able to replicate (REPLICATION)

2.     Should be chemically & structurally STABLE.

3.     Scope for slow change/Mutation (required for Evolution)

4.     Should be able to express itself in “Mendelian Characters”.

Ø   Due to base pairing both (DNA & RNA) can direct their replication. Protein fails to do so.

Ø STABILITY:- (a) Complementary strands of DNA, if separated by heating, reunites under proper conditions. (b) the 2’-OH group present on RNA is a reactive group (makes RNA labile & degradable). (c) RNA is also known to be CATALYTIC, hence reactive. (d) Presence of Thymine instead of Uracil (advantage during DNA repair).

Ø RNA Advantage:- RNA being unstable, mutate faster (Virus evolves faster). RNA can directly code for Protein Synthesis, DNA depends on RNA polymerase & its machinery.

Ø Both DNA & RNA act as genetic material. DNA being more stable, is preferred for STORAGE of genetic information, while RNA for TRANSFERRING them.

    Ribo -Nucleic Acid (RNA):-RNA was the first Genetic Material.

    “Replication of DNA” – Watson & Crick (1953).

   EXPERIMENTAL PROOF FOR REPLICATION OF DNA


 

(Meselson & Stahl, 1958)

                                                        

1.     They grew E.coli in ¹⁵NH₄CI medium (heavy isotope of Nitrogen) as the only source of Nitrogen, for many generations (¹⁵N incorporated in the newly synthesized DNA & other Nitrogen containing compounds).

2.     Heavy DNA & light DNA can be distinguished by centrifuging in a Cesium Chloride (CsCl) density Gradient (as ¹⁵N is not a radioactive isotope & can be separated from ¹⁴N by density gradient centrifuge).

3.     Then the bacterial cells were transferred to a medium with light ¹⁴NH₄Cl & took samples at regular intervals as the cells multiplied, and extracted the DNA to measure the density on CsCl gradient.

4.     E.coli divides every 20 min. Hence, second generation onwards, DNA was found to be HYBRID between the two DNA’s with intermediate density. After another generation (40 min.) it was composed of equal amounts of Hybrid & Lighter DNA’s.  {Next generation (60 min.) with 75% hybrid & 25% Light}.

5.     CONCLUSION: - DNA replication is Semi – Conservative.

                                                [ VIDEO OF MESELSON & STAHL'S EXPERIMENT ]

6.       A similar experiment using radioactive THYMIDINE to detect distribution of newly synthesize DNA in the chromosome was performed on Vicia faba (Faba Beans) {Tylor et al., 1958} proved that DNA on chromosomes also replicate semi- conservatively.

REPLICATION MECHINARY & THE ENZYMES: -

ü The replication requires a set of enzymes (main is DNA dependent DNA-Polymerase. As it uses a DNA template)

ü Highly Efficient enzymes (E.coli has 4.6 X 10⁶bp which are replicated in 38 minutes. i.e.. 2000 bp / second)

ü Deoxyribonucleoside Triphosphates have DUAL FUNCTION. Act as Substrate for polymerization reaction in replication & Provides energy for the process (like ATP).

ü For long DNA molecules, as two strands can’t be separated in entire length ( due to very high energy requirements) the replication occurs at small openings (REPLICATION FORK).

ü DNA polymerase catalyze polymerization only in 5’ → 3’ direction, hence one strand with polarity 3’ → 5’ replicates CONTINUOUS while the other strand (5’ → 3’) replicates in small fragments (OKAZAKI Fragments) & are joined later by DNA Ligase (DISCONTINUOUS Synthesis).

ü Origin of Replication (Ori): - Replication starts at specific sites called Ori. DNA polymerase cannot start the process of Replication itself.

(In Eukaryotes, Replication take place at S-phase of Cell Cycle.)

                                               [ VIDEO OF DNA REPLICATION ]

    TRANSCRIPTION

( Process of copying Genetic information from one strand of DNA in to RNA)

1.     Transcription follows the principle of complementarity (except, here A = U and not with T).

2.     Unlike replication (which once start, duplicates the total DNA) Transcription is segment specific (TEMPLET) from initiation to termination (start to finish).

3.      BOTH STRAND NOT COPIED: - (a) if both strand synthesize RNA, they code for different protein which will complicate genetic machinery. (b) Two simultaneously produced RNA will be complementary & will join to form double stranded RNA & no translation of protein will be done.

4.     DNA dependent RNA - polymerase also catalyze polymerization in 5’ → 3’ direction. ( DNA strand with 3’ → 5’ polarity is TEMPLET STRAND & opposite one is CODING STRAND as the RNA synthesized is having same code except for U in place of T).

5.     The PROMOTER (Towards 5’ end/Upstream) & TERMINATOR (3’ end/Downstream) are present on either sides of STRUCTURAL gene in the Transcriptional Unit.

6.     A DNA sequence (REGULATOR) near promoter provides binding for RNA - Polymerase.

7.     A gene coding for Structural unit/ polypeptides is called a CISTRON, only one (MONOCISTRONIC eg. Eukaryotes) or many (POLYCISTRONIC.eg. Prokaryotes).

8.     Eukaryotic Cistron has interrupted coding sequences (SPLIT GENES).  Coding/Expressed sequences (EXONS) are interrupted by non coding/intervening sequences (INTRONS). Exones are the sequences that appear in matured/processed RNA.

Ø MECHANISM OF TRANSCRIPTION (PROKARYOTES)

1.     Bacteria have three major types of RNA: - mRNA, tRNA & rRNA. (Messenger/transfer/ribosomal). All required in protein synthesis.

2.     mRNA provides template/Code, tRNA Anticodon (decoding) & rRNA play structural & catalytic role.

3.     Single DNA dependent RNA – Polymerase catalyze all types of Transcription in Prokaryotes.

4.     INITIATION: - RNA polymerase binds to INITIATION FACTOR (sigma) at the promoter & Initiates Transcription, using Nucleoside Triphosphate as substrates (ATP/GTP/CTP/UTP) following the rule of Complementarity.

5.     ELONGATION: - Binding of RNA polymerase to promoter causes opening of DNA helix & chain elongation.

6.     TERMINATION: - When RNA polymerase reaches the Termination region where a Terminator Factor (Rho) is present, it detaches from the DNA template & nascent RNA is released.

7.     In Bacteria, mRNA doesn’t require any processing & hence immediately becomes active.

8.     Sometimes, Translation starts even before the completion of Transcription is completed.

Ø MECHANISM OF TRANSCRIPTION (EUKARYOTES)

1.     EUKARYOTES have three major types of RNA: - mRNA, tRNA & rRNA. (Messenger/transfer/ribosomal). All required in protein synthesis.

2.     mRNA provides template/Code, tRNA Anticodon (decoding) & rRNA play structural & catalytic role.

3.     THREE RNA – POLYMERASES (I/II/III): - Three Polymerases in nucleus in addition to that found in organelles. RNA Polymerase-I transcribes RNA’s (28S, 18S and 5.8S), RNA Polymerase-II transcribe precursors of mRNA (hnRNA/heterogeneous nuclear RNA) and RNA Polymerase-III is responsible for Transcribing {tRNA, 5srRNA & snRNA (small nuclear RNA)}.

4.     INITIATION: - RNA polymerase binds to INITIATION FACTOR (σ-sigma) at the promoter & Initiates Transcription, using Nucleoside Triphosphate as substrates (ATP/GTP/CTP/UTP) following the rule of Complementarity.

5.     ELONGATION: - Binding of RNA polymerase to promoter causes opening of DNA helix & chain elongation.

6.     TERMINATION: - When RNA polymerase reaches the Termination region where a Terminator Factor (ρ-Rho) is present, it detaches from the DNA template & nascent RNA is released.

7.     SPLICING: - Primary Transcripts are non functional containing EXONS & INTRONS. Introns are removed & Exons are joined in a defined order to make it functional.

8.     hnRNA undergoes CAPPING (an unusual nucleotide eg. Methyl Guanosine Triphosphate/^mG_ppp is added to 5’ end) & TAILING (adding 200-300 Adenylated Residue/ Poly A sequence/ AAA…AA at 3’ end). This fully processed hnRNA is now called mRNA, is transported out of nucleus for Translation.

                                   [ VIDEO OF TRANSCRIPTION IN PROKARYOTES ]

                                   [ VIDEO OF TRANSCRIPTION IN EUKARYOTES ]

    GENETIC CODE: -

                      [George Gamow]

    George Gamow explained the triplet nature of codon. As only 4 bases codes for 20 amino acids, a triplet will generate 4³ (4x4x4) or 64 codons (many more than required).

    Dr. Hargobind Khorana CHEMICALLY synthesized Homopolymers & Copolymers of codons  for RNA.

                               [Dr. Hargobind Khorana]

    Marshall Nirenberg’s cell free system for protein synthesis finally helped to decipher the code.

                                                   [Marshall Nirenberg]

    Severo Ochoa enzyme (Polynucleotide Phosphorylase)was also helpful in polymerizing  RNA with defined sequences in a template independent manner (enzymatic synthesis of RNA).

    SALIENT FEATURES OF GENETIC CODE: -

1.     The Codon is TRIPLET.  61 codons code for amino acid& 3 for nothing (hence NONSENSE/ stop Codon).

2.     Any one codon codes for only One amino acid (UNAMBIGUOUS & SPECIFIC) eg. AUG for Methionin.

3.     DEGENERATE: - Some amino acids are coded by more than one codon, due to WOBBLE position of bases (last base decides the nature) eg. GUU/GUC/GUA/GUG all codes for VALINE. Similarly, UUU/UUC for Phenyl alanin & UUA/UUG for Leucine (first two bases common-UU) .

4.     Codons in RNA are read in continuous fashion without any Punctuation. Eg  --Val—Ala—Arg—Gly—Glu--  are expressed as – GUGGCCCGCGGUGAA-- .

5.     Nearly UNIVERSAL. Eg. In all organisms UUU codes for Phenylalanine (Phe). Some exceptions found in Mitochondrial DNA & some Protozoans.

6.     DUAL FUNCTION OF AUG :- Codes for Methionine (Met) & functions as INITIATION Codon.

                                                 [ VIDEO: FEATURES OF GENETIC CODE ]

        MUTATIONS & GENETIC CODE: -

Ø FRAME SHIFT MUTATION (Addition/Insertion or Deletion of one or more  bases shifts the sequence)

Ø Addition/Insertion of bases shift the frame towards RIGHT (→) eg. AUG UAC AAG GUA  reads as –Met—Try—Lys – Val --  but if addition of, let’s say  ‘A’ is done at 4^th position, then AUG AUA CAA GGU A—  will read as  -- Met – Ile—Gin—Gly--. Hence the whole nature of protein is changed.

Ø Deletion of a base shifts the frame towards LEFT (←) eg. AUG UAC AAG GUA  reads as –Met—Try—Lys – Val --  but if the 5^th base ‘A’ is deleted, the sequence will be AUG UCA AGG UA_  which will be expressed as –Met – Ser – Arg – Try / Stop Codon.

Ø SUBSTITUTION:- eg. Sickle Cell Anaemia is caused by substitution of Glutamic Acid (GAG) by Valine (GUG) at the 6^th position of second Beta-Globin chain of Hemoglobin.

                             [ VIDEO: UNDERSTANDING MUTATION & ITS TYPES ]

    tRNA (The ADAPTER MOLECULE): -

1.     Francis Crick postulated the presence of an Adapter Molecule that, on One hand read the codon and on other hand would bind to specific amino acids.

2.     Though tRNA was known much earlier before the Genetic Code was postulated (then called Soluble RNA or sRNA), its role as an adepter molecule was assigned much later.

3.     tRNA are specific to each amino acid. Eg. Initiator tRNA for Methionin (Met) coded by AUG in mRNA.

v CLOVER LEAF STRUCTURE (2D-structure): -

1.     This structure has 4 arms [DHU loop (7-8 bases), The Anticodon (7-bases), TφC loop (7-bases) and sometime a Variable Arm/ The Lump (3-5 bases)].

2.     5’-Terminus has Poly P sequence & 3’-End has CCA-OH Terminus (Amino acid attachment site).

3.     ANTICODON LOOP has 3 bases (ANTICODON) complementary to mRNA codons.

4.     Clover leaf structure is a Modal while the actual 3D structure of tRNA is like an inverted L.

    TRANSLATION

(Process of polymerization of amino acids to form a polypeptide through Codon-Anticodon)

1.     The order & sequence of amino acids in the polypeptide is based on the sequence of bases in the mRNA.

2.     Amino acids are joined by PEPTIDE BONDS (endothermic reaction). ATP is required for this.

3.     Activation of Amino Acids in the presence of ATP ( AA + ATP → AA*).

4.     CHARGING/AMINIACYLATION OF tRNA: - AA* + tRNA → AA*~tRNA (specific bonding)

5.     Cellular factory responsible for Protein Synthesis are a Structural RNA & 80 different proteins.

6.     INITIATION: - AA*~tRNA + 40S (small subunit Ribosome) → AA*~tRNA~40S Complex.  This complex initiates the Translation process. First codon (AUG) is read by special INITIATOR tRNA only.

7.     CHAIN ELONGATION: -   AA*~tRNA~40S + 60S (large subunit of Ribosome) → AA*~tRNA~RIBISOME. There are Two Sites on Larger Subunit of ribosome, one for attachment of amino acid (A-site) & other for formation of Polypeptide chain by peptide bond formation (P-Site).  Ribosome also acts as Catalyst for peptide formation eg. 23S RNA in Bacteria is the enzyme (RIBOZYME).

8.     CHAIN TERMINATION: - As the complex reaches the STOP/NONSENSE CODON (UAA/UAG/UGA) which does not codes for any codon (not recognized by any tRNA, a RELEASE FACTOR binds to it and terminates the synthesis. The chain is released in the TUNNEL region of Ribosome.

9.     UNTRANSLATED REGIONS (UTR’s): - An mRNA also contains UTR’s, both at 5’-end (before AUG) & at 3’-end (After Stop Codon) for efficient Translation Process (Capping & Tailing).

[ VIDEO OF TRANSLATION IN PROKARYOTES ]

[ VIDEO OF TRANSLATION IN EUKARYOTES ]

    REGULATION OF GENE EXPRESSION : -

Ø Gene Expression is regulated at various level:

 (a) Transcription (formation of Primary Transcript)

(b) Level of Processing (Splicing)

 (c) Transport of mRNA from nucleus to cytoplasm 
(d) Translation level.

OPERON CONCEPT:

                                                                      (Jacob & Monod)

Ø It is the metabolic/physiological/environmental conditions that regulate the gene expression. Eg. In Lac-Operon, Gene expression converts Lactose in to Glucose & Galactose which Bacteria use for energy. If no Lactose is present in the surrounding, no gene expression will be done.

Ø In Prokaryotes, site of Transcriptional Initiation is the predominant site for Gene Regulation. It could be Positive (Activators) or Negative (Repressors/Inhibitors).

Ø OPERATOR (O) Region adjacent to PROMOTER (P) Region, mostly binds to Repressor Protein.

Ø OPERON is a Polycistronic gene regulated by a common promoter & Regulator gene. Common in Bacteria eg. Lac-Operon, try-Operon, ara-Opreon, his-Operon, val-Operon etc..

    THE Lac-OPERON: - Negative Feedback Regulation Mechanism.

1.     It regulates the conversion of Lactose in to Glucose & Galactose for energy production in bacteria.

2.     It Consists of a Regulatory gene (i- which is derived from the word Inhibitor/repressor not inducer), Three Structural genes (z, y, a), Promoter (p) & an Operator (o).

3.     Three structural genes codes for different enzymes: - ‘z’ (β-Galactosidase) hydrolyse Lactose into Glucose & Galactose, ‘y’ (Lactose Permease) increase permeability of the cell to β-Galactosidase & ‘a’ for Transacetylase which acylates the product.

4.     Presence of Lactose switches on the lac-Operon (Lactose as INDUCER). So, lactose in the medium is  transported in to the cell by Permease & acted upon by β-Galactosidase to produce simple sugar.

5.     The REPRESSOR is synthesized continuously from gene ‘i’ which bind to the operator & prevent RNA-Polymerase from transcribing Operon. (SWITCH OFF).

6.     Inducer (eg. Lactose, Allolactose) binds to the repressor & inactivates it. (SWITCH ON)
                                 [ VIDEO OF Lac OPERON ]

            THE HUMAN GENOME PROJECT (HGP): -

ü Launched in 1990 as a MEGA PROJECT. Human genome has approx. 3 x 10⁹ bp. The cost of sequencing is US $ 3 per bp (total cost US $ 9 billion). Further huge data storage required from a single cell, required fast computing devices for data storage & retrieval for analysis.

ü HGP was a13 year project completed in 2003, coordinated by US dept. of Energy & Health, WeIlcom Trust (UK) etc..

ü Sequencing of Chromosome No-1 was completed on May 2006 (last to be done)

ü BIOINFORMATICS: - Branch of biology where data storage in computers and sharing is done systematically.

ü OTHER GENOME PROJECTS: - Bacteria, Yeast, Drosophila, Plants (Rice & Arabidopsis) , a free living on pathogenic nematode (Caenorhabditis elegans) etc..

    GOALS OF HGP: -

1.     Identify all genes (20,000 – 25,000) & Determine the sequences of 3 billion bp in human DNA.

2.     Storage of Information  in Data base & improve tools for Data Analysis.

3.     Transfer related technologies to other sectors like Industry.

4.     Address Ethico-Legal & Social Issues (ELSI) arising from the Project.

    METHODOLOGY: - (Two Approaches)

Ø Identifying all genes that are expressed as RNA (Expressed Sequence Tags/ESTs).

Ø Blind approach of sequencing whole genome containing coding & non coding sequences (SEQUENCE ANNOTATION). Here, The total DNA from the cell is isolated, randomly fragmented in smaller sizes (for technical reasons) & cloned in suitable hosts to amplify each piece of DNA for sequencing ease. Common Vectors used were Bacterial Artificial Chromosome (BAC) & Yeast Artificial Chromosome (YAC).

                                      [ VIDEO EXPLANATION IN HINDI ]

Ø The fragments were sequenced using automated DNA sequencing (SANGER METHOD). These sequences were then arranged based on Overlapping regions present in them (done by generating overlapping fragments for sequencing). Alignment of these sequences was done based on Computer Programming.

[ VIDEO OF SANGER METHOD ]

Ø Genetic & Physical maps on genome were assigned using information on polymorphism of Restriction Endonuclease recognition sites & repetitive DNA sequence (Microsatellite).

       SALIENT FEATURES OF HUMAN GENOME: -

1.     Contains 3164.7 million nucleotide bases.

2.     A Gene contains ~3000 bases but size vary greatly (largest known Human gene is DYSTROPHIN with 2.4 million bases.

3.     Total number of Genes ~ 30,000. Almost 99.9% of bases are Exactly same in all humans.

4.     50 % of discovered genes have Unknown functions. Less than 2 % code for Proteins.

5.     Repeated Sequences of DNA  make up of very large number of Human Genome. They have no direct coding functions but shed light on Chromosomal Structure, Dynamics & Evolution.

6.     Chromosome No. 1 has most genes (2968) & Y-chromosome has least (231).

7.     SNIPS: - (SINGLE NUCLEOTIDE POLYMORPHISM or SNPs) pronounced as ‘”NIPS”.  About 1.4 million locations where single base differences occurs in human. Snips  have significance in tracing chromosomal locations for disease associated sequences & tracing Human History.[ VIDEO ]

    APPLICATION & FUTURE CHALLENGES: -

Ø Enable Biological research with a whole new radical approach. Whole genome coupled with new Technology will enable us to approach questions & challenges in a more systematic & broader scale.

    DNA FINGERPRINTING: -

Ø It involves identifying differences in some specific REPETITIVE Sequences (separate from bulk Genome as different ‘Peaks’ during Density Gradient Centrifugation). The bulk DNA forms a major peak & the other small peaks are known as SATELLITE DNA.

Ø Depending upon base composition (A:T rich or C:G Rich), length of satellite, number of repetitive units, the satellite is classified as Macro-Satellite, Mini-Satellite etc..[ VIDEO ]

Ø Satellite do not code for any Protein but show a high degree of Polymorphism & form the basis of DNA Fingerprinting. And hence Forensic Science.

Ø DNA Polymorphism (eg. Alleles): - Predominantly due to inherited mutation (Germinal Mutation).  The probabilities of such variations are high in Non Coding Sequences as they do not express. For studies on Evolution & Speciation such polymorphism are important (as the variations keep on accumulating generations after generations).

Ø VNTR (Variable Number of Tandem Repeats):- Satellite DNA with very high degree of Polymorphism.

    TECHNIQUE OF DNA FINGERPRINTING

                                                                       ( Sir Alec Jeffrey’s)

ü It used Satellite DNA with very high degree of polymorphism (Variable Number of Tandem Repeats or VNTR).[VIDEO]

ü The Technique used SOUTHERN BLOT Hybridization using Radiolabelled VNTR as Probes. It includes: -

1.     Isolation of DNA.

2.     Fragmentation of DNA (Digestion) by Restriction Endonuclease.

3.     Separation of DNA fragments by ELECTROPHORESIS.[VIDEO]

4.     Transferring (BLOTTING) of separated DNA fragments to Synthetic Membranes. Eg. Nitrocellulose, Nylon rtc..

5.     Hybridization using labeled VNTR probe.

6.     Detection of Hybridized DNA using Autoradiography

ü  A small DNA is arranged tandemly in many copy numbers.

ü  The copy number varies from chromosome to chromosome in an individual. The number of repeat show very high degree of polymorphism.

ü  As a result the size of VNTR varies from 0.1 to 20kb. So, after Hybridization with VNTR probes, the autoradiograms give many bands of different sizes. (Characteristic of a Person).

                                               [ VIDEO OF DNA FINGERPRINTING PROCESS ]

ü  Exception is Monozygous Twins (Identical Twins).

ü  The sensitivity of technique was increased by using Polymerase Chain Reaction (PCR) technique. (producing bulk DNA). [VIDEO]

ü  SIGNIFICANCE: - Forensics, Population & Genetic Diversities.

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