NUCLEIC ACID GENE IS THE ELEMENT CONTAINING INFORMATION THAT IS HANDED DOWN TO DAUGHTER CELLS (NUCLEIC ACID)GENE CAN BE IN THE FORM OF DNA OR RNA (NUCLEIC ACID) DNA DISCOVERY FRIEDERICH MIESCHER CALLED IT NUCLEIN BECAUSE IT CAN BE FOUND IN THE NUCLEUS AND ASSUMED TO BE ON THE CHROMOSOME

NUCLEIC ACID HOW WAS DNA KNOWN TO BE THE GENETIC MATERIAL? 1944-Oswald Avery, Colin MacLeod and Maclyn McCarty -DNA and not protein or RNA is the material that transforms bacteria (experiment using Streptococcus pneumoniae inoculated to mice) Alfred Hershey and Martha Chase - DNA is the molecule inherited to bacteriophage T4

TRANSFORMATION

THE HERSHEY-CHASE EXPERIMENT

THE CHARGAFF COMPLIMENTARY RELATIONSHIP RULE DNA from a different species have the same number of thymine and adenine; the number of guanine and cytosine is also the same (A=T)/(G=C) pair of one purine and one pirimidine is in the ratio of 1.00

THE WATSON-CRICK MODEL A MODEL THAT EXPLAINS THE STRUCTURE OF DNA (1953) GATHERED FROM THE INFORMATION ON CHEMICAL DATA ANALYSIS (BASE COMPOSITION-THE CHARGAFF RULE) AND PHYSICAL (X-RAY DIFRACTION ANALYSIS - INFORMATION ON THE DIMENSION AND THE ARRANGEMENT OF DIFFERENT PARTS OF MOLECULES) CLASSIC MODEL (Watson & Crick) X-RAY DIFRACTION ANALYSIS (Rosalind Franklin & Maurice Wilkins)

THE WATSON-CRICK MODEL MINOR GROOVE MAJOR GROOVE *SUGAR-PHOSPHATE BACK-BONE (OUTER) * NITROGEN CONTAINING BASE PAIR (INNER) A+T OR G+C 34Å 3.4Å 20Å *2 ANTI-PARALLEL DNA CHAIN *COMPLIMENTARY TO EACH OTHER *BASE PAIRS BONDED BY HYDROGEN BONDS

DNA STRUCTURE BASE COMPOSITION ANTI-PARALLEL ORIENTATION - 5’-PHOSPHATE END (5’-P) AND 3’- HYDROXYL END (3’-OH) ARE AVAILABLE AT THE ENDS OF BOTH CHAINS -THE SUGARS ARE IN DIFFERENT POSITIONS; FACING UP AND DOWN, AN IMPORTANT POSITION DURING REPLICATION PROCESS THE ATC SEQUENCE OF A DNA CHAIN IS WRITTEN AS-P-5’-ATC-3’-OH OR pApTpC

DNA STRUCTURE THE DIVERSITY IN BASE COMPOSITION OF DNA IN DIFFERENT ORGANISME -[A]=[T] AND [G]=[C] - RATIO([G]+[C]) ([A]+[T]) RATIO RANGING FROM 0.37 TO G+C CONTENT OR G+C PERCENTAGE = G+C G+C +A+T - HAVING CLOSE RELATIONSHIP WITH DNA DENSITY

DNA STRUCTURE FORMS OF DNA DOUBLE HELIX -RIGHT DIRECTION: CLOCKWISE TURN (B/A- FORM) - LEFT DIRECTION: ANTI-CLOCKWISE TURN (Z- FORM) B FORM: NATURALLY OCCURING A FORM: NATURALLY OCCURING (AT CERTAIN SITES) -SYNTHETIC DNA/dsRNA/DNA:RNA PAIR Z FORM: IN VITRO CONDITION- HIGH SALT CONTENT

DNA STRUCTURE FORMS THAT COULD BE SEEN VIA THE X-RAY DIFRACTION ANALYSIS DATA: - A-FORM (RELATIVE HUMIDITY 66%)-11bp - B -FORM (RELATIVE HUMIDITY 92%)-10bp - Z -FORM (12bp) (PER HELIX TURN)

DNA STRUCTURE THE SIZE OF DNA MOLECULE MOLECULAR WEIGHT (MW) PER UNIT HELICAL LENGTH IS 2X10 6 PER MIKROMETER (  m) e.g.: MW OF BACTERIAL DNA 1mm LONG IS 2X10 9 WIDTH 20Å =  m LENGTH BETWEEN 2 BASES 3.4Å =  m LENGTH OF A HELICAL TURN=  m FAJ T7 (MW=25X10 6 ), FAJ T4 (MW=106X10 6 ) BACTERIA (MW= X10 9 ), YEAST (MW=6X10 8 ), HUMAN (MW=8X10 10 )

RNA STRUCTURE COMPOSITION IN CELL: 10X MORE THAN DNA THE GENETIC MATERIAL FOR CERTAIN VIRUSES SINGLE STRANDED POLINUCLEOTIDE 4 IMPORTANT TYPES: a) RIBOSOMAL RNA (rRNA) b)TRANSPORT RNA (tRNA) c) MESSENGER RNA (mRNA) d) EUCARYOTIC RNA MOLECULE - RIBONUCLEOPROTEIN PARTICLE

RNA STRUCTURE

PRECURSOR TO RNA SYNTHESIS - 4 RIBONUCLEOSIDE 5’-TRIPHOSPHATE (NTP): - ATP, GTP, CTP, UTP POLIMERISATION OF 3’-OH OF ONE NUCLEOTIDE TO THE 5’-TRIPHOSPHATE OF ANOTHER NUCLEOTIDE TO FORM A PHOSPHODIESTER BOND THE SEQUENCES IS DETERMINED BY DNA SEQUENCES IT IS TRANSCRIBED FORM SINGLE STRANDED DNA THAT ACTS AS A TEMPLATE 5’-PPP 5’ 3’ 3’-OH

RNA STRUCTURE ADENINE (A) ADENOSINE ADENYLATE GUANINE (G) GUANOSINE GUANYLATE CYTOSINE (C) CYTIDINE CYTIDILATE URACIL (U) URIDINE URIDILATE NUCLEOTIDE COMBINATION NUCLEOSIDE COMBINATION BASE

FACTORS AFFECTING DNA STRUCTURE DENATURATION- -A CONDITION IN WHICH DNA CONFORMATION IS DISTURBED OR ALTERED FROM THE NATURAL FORM -DNA DOUBLE HELIX UNWINDS RENATURATION- -DENATURATED DNA RENATURE INTO ITS NATURAL FORM -DNA DOUBLE HELIXS RE-FORMS)

DENATURATION THE H BOND OR HYDROPHOBIC INTERACTION BETWEEN CHAINS IS DISTURBED TO CAUSE DNA TO SEPARATE INTO ssDNA * HEAT * CHEMICALS-DENATURING AGENTS THE METHOD TO DETERMINE DISRUPTION OF DNA/ TO DNA QUANTIFICATION : OPTICAL ABSORBANCE AT 260NM (C) dsDNA A 260 =1.00 ssDNA A FREE BASES A 260 =1.60

DENATURATION HEAT OR CHEMICAL TREATMENT: MELTING CURVE - A PLOT OF OD 260 VS EITHER TEMPERATURE OR CONCENTRATION OF A DENATURING REAGENT; LOSS OF ORDERED STRUCTURE OCCURS Conc.

DENATURATION FOR THERMAL DENATURATION: MELTING TEMPERATURE Tm: THE TEMPERATURE AT WHICH THE INCREASE IN OD 260 IS 50% OF THAT REACHED WHEN STRAND SEPARATION IS COMPLETE Conc.

DENATURATION THE DECREASE IN Tm VALUE CAN OCCUR WHEN DNA IS EXPOSED TO: A) CHEMICALS THAT CAN FORM HYDROGEN BONDS WITH NUCLEOTIDES e.g.: UREA, FORMAMIDE B) CHEMICALS THAT CAN INCREASE THE INTERACTION BETWEEN AND WATER SUCH AS METANOL C) CHEMICALS THAT CAN WEAKENS HYDROPHOBIC INTERACTION SUCH AS SODIUM TRIFLUOROACETATE

DENATURATION IN THE DNA STRUCTURE, ELECTROSTATIC INTERACTION OCCURS BETWEEN THE NEGATIVELY CHARGED PHOSPHATE (INTER CHAIN) THE CHAINS TEND TO PUSH EACH OTHER IF THE CHARGES ARE NOT NEUTRALISED Tm VALUE WILL DECREASE WHEN DNA IS SOLUBILISED IN A DECREASING CONCENTRATION OF SALT SOLUTION e.g (Na + )-PO 4 - DNA WILL DENATURE IN WATER AT ROOM TEMPERATURE

DENATURATION CHEMICALS THAT CAN DECREASE HYDROPHOBIC INTERACTION CAN ALTER THE BASE SEQUENCES HEATED DNA WILL CAUSE BASES STACKING TO DECREASE AND THEREFORE INCREASES OD 260 CHEMICALS THAT BREAK HYDROGEN BONDS DOES NOT AFFECT BASES STACKING OF ssDNA BUT ONLY dsDNA H-BOND AND BASE STACKING STABILISED DNA STRUCTURE

DENATURATION EFFECTS OF ALKALI ON DNA HIGH pH WILL ALTER THE CHARGED GROUPS THAT ARE INVOLVED IN H-BOND H-BOND CANNOT OCCUR AT pH > 11.3 AND DNA DENATURES THE MOST COMMON METHOD TO DENATURE DNA SINCE DNA IS QUITE RESISTANT TO HYDROLISIS

DENATURATED DNA STRUCTURE DENATURATION COMPLETES AT 90  C WITH THE FORMATION OF ssDNA AND UNSTACKED BASES IF THE TEMPERATURE IS LOWERED TO ROOM TEMPERATURE WITH THE PRESENCE OF SALT NOT MORE THAN 0.05M, H-BOND CAN FORM BETWEEN SINGLE STRANDS TO FORM A STABLE STRUCTURE IN THE PRESENCE OF LOW SALT CONCENTRATION (<0.01M), ssDNA DOES NOT FORM H-BOND BECAUSE OF THE PHOSPHATE GROUPS

DENATURATED DNA STRUCTURE THE REFORMATION OF H-BOND BETWEEN DNA STRANDS NEEDS HIGH DNA AND SALT CONCENTRATION HIGH SALT LOW SALT

RENATURATION TWO IMPORTANT CONDITIONS FOR RENATURATION TO OCCUR: SALT CONCENTRATION IS HIGH ENOUGH TO DECREASE THE ELESTROSTATIC INTERACTION OF THE PHOSPHATE GROUPS (0.15M-0.5M NaCl) TEMPERATURE IS HIGH ENOUGH TO PREVENT RANDOM H-BONDING BUT ALLOWS STABLE BASE PAIRING BETWEEN STRANDS, TEMPERATURE 20-25°C BELOW THE OPTIMUM Tm

MECHANISM 1A1BII1C...ATGA….ATGA….CCCC…..ATGA…. …..TACT….TACT….GGGG…..TACT…. 1A’1B’II’1C’ SHORT SEQUENCES OF 4-6 BASES CAN OCCUR SEVERAL TIMES IN A DNA MOLECULE PAIRING OF SEQUENCE 1A AND II’ IS IMPOSSIBLE BUT NOT 1A & 1B’/1C’. HOWEVER PAIRING WILL NOT LAST DUE TO NO STABILISATION BY BASE STACKING

HYBRIDISATION RENATURATION: THE PRINCIPLE BEHIND HYBRIDISATION PAIRING OF 2 SINGLE STRANDS OF DNA/RNA COMPLIMENTARY TO EACH OTHER USING NITROCELLULOSE MEMBRANE AS THE SUPPORT TO HOLD THE SUGAR-PHOSPHATE BACKBONE AND THE BASES ARE EXPOSED

FACTORS AFFECTING RENATURATION TWO SINGLE STRANDS MUST FIRST COLLIDE WITH EACH OTHER LONG TRACTS OF BASES MUST BE IN PROPER REGISTER TO ALLOW COMPLETE RENATURATION RENATURATION RATE INCREASE WITH DNA CONCENTRATION FAJ T7 (MW=2.5X10 7 ), FAJ T4 (MW=1.1X10 8 ) t 1/2 T7< t 1/2 T4, t 1/2 = HALF OF THE TIME NEEDED FOR THE RENATURATION PROCESS TO COMPLETE

FACTORS AFFECTING RENATURATION HETERODUPLEX: -BASE PAIRING BETWEEN 2 STRANDS WITH NON-COMPLIMENTARY REGIONS -IF HIBRIDISATION OCCURS, THE NEW STRAND WILL HAVE DELETED SEQUENCES

VARIANTS OF DNA STRUCTURE CIRCLE: THE TWO ENDS OF A LINEAR DNA ARE COVALENTLY JOINED, A CIRCLE CONTAINING TWO CONTINUOUS STRANDS : COVALENTLY CLOSED CIRCLE (CCC)

VARIANTS OF DNA STRUCTURE SUPERCOILED OR SUPERHELIX DNA: THE TWO ENDS OF A LINEAR DNA ARE ROTATED BEFORE JOINING AND THEN FORMING A COVALENT CIRCLE, THE RESULTING MOLECULE WILL BE TWISTED CIRCLE

VARIANTS OF DNA STRUCTURE NICKED CIRCLE: -IF ONE OR MORE SINGLE-STRAND BREAKS ARE PRESENT IN A CIRCLE, THE MOLECULE IS CALLED A NICKED CIRCLE OR AN OPEN CIRCLE

VARIANTS OF DNA STRUCTURE SINGLE STRANDED TERMINI: THE DNA OF THE E. coli PHAGE HAS A SINGLE STRANDED PROJECTION 12 BASES LONG AT EACH END OF THE DNA. THE BASE SEQUENCES ARE COMPLEMENTARY SO THAT THE SINGLE STRANDS CAN FORM A DOUBLE-STRANDED SECTION TO CONVERT THE LINEAR DNA TO A CIRCLE: COHESIVE SITES OR STICKY ENDS. A CIRCLE FORMED IN THIS WAY IS CALLED A HERSHEY CIRCLE.

VARIANTS OF DNA STRUCTURE UNUSUAL BASES -BASES OTHER THAN A, G, T AND C FOUND IN DNA MOLECULE OF SOME TYPES OF PHAGES; U REPLACES T, 5’HYDROXYMETHYLCYTOSINE (HMC) REPLACES C # GLUCOSYLATED HMC IN E. coli PHAGES (protection from phage-induced nucleases) # METHYLATED ADENINE, METHYLATED CYTOSINE- (part of host restriction system)

SPECIAL BASE SEQUENCES REPEATED SEQUENCES (PALINDROME) A B C D E E’ D’C’B’A A’B’C’D’E’E D C B A REVERSED SEQUENCES, REVERSED REPEATS ‘CRUCIFORM’ STRUCTURE: ‘HAIRPIN’ OR ‘STEM AND LOOP’