1. NUCLEOTIDES

2. Nucleic acids
Nucleic acids are polymers of monomers called nucleotides
2 types of nucleic acid – DNA & RNA
Each nucleotides composed of 3 parts :
- A nitrogenous base pyrimidines
purines
- A pentose (5-C sugar)
- Phosphate group

3. Nitrogenous base

4. A, G, C found as both RNA and DNA
U is found as RNA
T is found as DNA

5. PENTOSE SUGAR In ribonucleotides, the pentose is ribose
In deoxyribonucleotide (or deoxynucleotides) the sugar is 2’-deoxyribose – the carbon at position 2’ lacks a hydroxyl group

7. In a nucleic acid polymer/polynucleotide, nucleotides are joined by covalent bonds = phosphodiester linkage (between phosphate of one nucleotides and the sugar of the next)

8. Nucleic acid structure
Nucleotides can be joined to each other to form RNA and DNA.
The nucleic acids are chains of nucleotides whose phosphates bridge the 3' and 5' positions of neighboring ribose units
The phosphates of these polynucleotides are acidic, so at physiological pH, nucleic acids are polyanions.
The linkage between individual nucleotides is known as a phosphodiester bond.

9. Each nucleotide that has been incorporated into the polynucleotide is known as a nucleotide residue.
The terminal residue whose C5' is not linked to another nucleotide is called the 5' end
The terminal residue whose C3' is not linked to another nucleotide is called the 3' end.
By convention, the sequence of nucleotide residues in a nucleic acid is written, left to right, from the 5' end to the 3' end.

10. Nucleic acid structure

11. Definitions
DNA stands for deoxyribonucleic acid. It is the genetic code molecule for most organisms.
RNA stands for ribonucleic acid. RNA molecules are involved in converting the genetic information in DNA into proteins. In retroviruses, RNA is the genetic material.

12. DNA structure : Watson and Crick
Watson and Crick st proposed the double helix as 3-D structure of DNA
Two polynucleotide chains wind around a common axis to form a double helix.
The two strands of DNA are antiparallel, but each forms a right-handed helix.
The bases occupy the core of the helix and sugar-phosphate chains run along the periphery, thereby minimizing the repulsions between charged phosphate groups.

13. DNA consist of 2 polynucleotide strands wound around each other to form a right-handed double helix

14. Nucleotides are linked each other by 3’-5’ phosphodiester bonds (join 5’-hydroxyl group of deoxyribose sugar of one nucleotide to the 3’-hydroxyl group of deoxyribose of another nucleotide)
Hydrogen bond form between the nitrogenous base of 2 antiparallel polynucleotide strands
TWO types of base pairs in DNA :
1) adenine (purine) pairs with thyamine (pyrimidine)
2) Guanine (purine) pairs with cytosine (pyrimidine)

15. If 1 strand has the base sequence AGGTCCG, so the other strand must have sequence TCCAGGC
These hydrogen­bonding interactions, a phenomenon known as complementary base pairing, result in the specific association of the two chains of the double helix.

17. Dimension of DNA :
1) one turn of double helix span 3.4nm consist 10.4 base pairs.
2) diameter of double helix is 2.4nm
3) distance between adjacent base pairs is 0.34nm.

18. Noncovalent bonding in DNA structure :
1) Hydrophobic interactions. Electron between stacked purine & pyrimidine bases is nonpolar. the clustering of bases component of nucleotide within double helix stabilize structure, because it minimize their interaction with water.
2) Hydrogen bond. Base pairs, on close approach form hydrogen bond, three between GC pairs and two between AT.

19. 3) Base stacking. Stacking interactions are a form of van der waals interaction. Interaction between stacked G and C bases are greater than those between stacked A and T bases, which largely accounts for the greater thermal stability of DNAs with a high G+C content
4) Electrostatic interaction. DNA external surface, sugar-phosphate backbone possesses –ve charged phosphate group.

20. The DNA helix The geometry of DNA
The biologically most common form of DNA is known as B-DNA, - structural features first noted by Watson and Crick together with Rosalind Franklin and other.
DNA is flexible molecule. It can assume several distinct structural depending on the solvent composition and base sequence. The major structural variants of DNA are A-DNA and Z-DNA.
Under dehydrating conditions, B-DNA undergoes a reversible conformational change to A-DNA which forms a wider and flatter right-handed helix than does B-DNA.

22. A-DNA When DNA become partially dehydrated, it assumes the A form.
The base pairs no longer at right angle
They tilt 20° away from the horizontal
Distance between adjacent base pairs slightly reduced (11bp helical turn instead or 10.4bp found in B form)
Each turn of double helix occur in 2.5nm, instead of 3.4nm
Diameter swell to 2.6nm

23. Z-DNA Named for it zigzag conformation
Diameter = 1.8nm, slimmer than B-DNA
Twisted into left-handed spiral with 12bp per turn
Each turn occur in 4.5nm

24. RNA Ribonucleic acid is a class of polynucleotides
In contrast, RNA occurs primarily as single strands, which usually form compact structures rather than loose extended chains
Nearly all involve in some aspect of protein synthesis
RNA molecules are synthesized in a process called TRANSCRIPTION
New RNA mol. are produced by mechanism similar to DNA, through complementary base pair formation

25. New RNA mol. Are produced by mechanism similar to DNA, through complementary base pair formation (A=U G=C)
Eg. DNA sequence 5’-CCGATTACG-3’ is transcribe into RNA sequence
3’-GGCUAAUGC-5’

26. Differences between DNA & RNA
Sugar moiety is ribose
Sugar moiety is deoxyribose
Nitrogenous base Adenine, Urasil, Guanine, Cytosine
Nitrogenous base
Adenine, Thyamine,
Guanine, Cytosine
Exist in single strand
Exist in double helix
Content of A and U, as well as G and C are equal
Content of A and T, as well as G and C are equal

27. Secondary structure of RNA
RNA exist as single strand.
RNA can coil back on itself and form a unique secondary structure
The shape of these structures determined by complementary base pairing by specific RNA sequence, as well as base stacking

28. Types or RNA Types of RNA = transfer RNA, ribosomal RNA, messenger RNA
tRNA transport amino acids to ribosomes for assembly into protein
Average length of tRNA = 75 nucleotides

30. Ribosomal RNA
rRNA is the most abundant RNA in living cells
rRNA is the component of ribosomes
Ribosomes = cytoplasmic structures that synthesized proteins
Messenger RNA
mRNA is the carrier of genetic information from DNA for the synthesis of protein
mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes

32. Denaturation and renaturation of DNA
Unique properties of nucleic acids- under certain conditions DNA duplexes reversibly melt (separate) and reanneal (base pair to form duplex again)
Binding forces that hold the DNA double helix can be disrupted
This process = denaturation, promoted by :
- heat (most common denaturing method)
- low salt concentrations
- extremes in pH

33. - Renaturation DNA can be prepared by maintain the temp.
below denaturing temp.
- requires some time because the strands
explore various configurations until they achieve the most
stable one

34. Nucleic acid methods
Most of technique used in nucleic acid research are based on differences in molecular weight or shape, base sequences, or complementary base pairing
Some of the most useful nucleic acid fractionation procedure are:
Chromatography
Electrophoresis
Ultracentrifugation

35. Nucleic acid extraction protocol
Ruptured bacterial cells or isolate eukaryotic nucleus
- to expose the nucleic acid
- done by grinding or sonicating the sample
Removing membrane lipids by adding a detergent or enzyme lysozyme
Removing proteins by adding a protease
Precipitating the DNA with an alcohol
- usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt

36. Chromatography
Many of the chromatographic techniques that are used to separate proteins also apply to nucleic acids
Objectives : purify nucleic acid of interest or isolation of individual nucleic acid sequences
A type of column chromatography that uses a calcium phosphate gel called hydroxyapatite been used in nucleic acid research
Hydroxyapatite bind tightly to double-stranded nucleic acid than single-stranded nucleic acid molecules
So dsDNA can be effectively separate from ssDNA, RNA or other protein contaminants by this method

37. Elute the column with a concentrated phosphate buffer tp collect dsDNA
dsDNA can be rapidly isolated by passing a cell lysate through a hydroxyapatite column
wash the column with a low concentration of phosphate buffer to release only the ssDNA, RNA and protein
Elute the column with a concentrated phosphate buffer tp collect dsDNA
RNA +
protein
hydroxyapatite
dsDNA

38. Affinity chromatography is used to isolate specific nucleic acids.
For example, most eukaryotic messenger RNAs (mRNAs) have a poly (A) sequences or cellulose to which poly (U) is covalently attached. The poly(A) sequences specifically bind to the complementary poly(U) in high salt and low temperature and can later be released by altering these condition.

39. Electrophoresis
Gel electrophoresis separate nucleic acids on the basis of molecular weight and 3-D structure in an electric field
The technique involves drawing DNA molecules, which have an overall negative charge, through a semisolid gel by an electric current toward the positive electrode within an electrophoresis chamber.
The used gel is typically composed of a purified sugar component of agar called agarose.

40. Electrophoresis Nucleuic acids mixture placed in well
Nucleic acids are -ve charge (phosphate group)
Nucleic acid migrate to anode
Rate of migration are proportional to molecular size

41. In genetic engineering, scientists use the technique to isolate fragments of DNA molecules that can then be inserted into vectors, multiplied by PCR, or preserved in a gene library.

42. Southern blotting
Enable researcher to detect and analyze particular DNA sequence
The basis of detecting specific sequence : nucleic acids hybridization
Hybridization can be used to locate and/ or identify specific genes or other sequence
Eg. ssDNA from two diff sources (tumor cell and normal cell) can be screened for sequence differences

43. Southern blott technique
1) restriction fragment preparation
DNA samples to be tested are treated with restriction enzymes that cut at specific nucleotides sequences to produce a restriction fragments
2) electrophoresis
The mixture of restriction fragments from each sample are separated by electrophoresis according to their size
Each sample forms a characteristic patterns of band
The gel soaked with 0.5M NaOH to convert dsDNA to ssDNA

44. Southern blot technique

45. 3) Blotting
The DNA fragments are transferred to nitrocellulose filter paper by placing them on a wet sponge in a tray with a high salt buffer (nitrocellulose bind strongly to ssDNA)
As buffer is drawn through the gel and filter paper by capillary action, the DNA is transferred and become permanently bound to nitrocellulose filter
4) hybridization with radioactive probe
Nitrocellulose filter is exposed to radioactively labeled probe, which bind to ssDNA with a complementary sequence

46. 4) hybridization with radioactive probe
Nitrocellulose filter is exposed to a solution containing radioactively labeled probe.
The probe is ssDNA complementary to DNA sequence of interest, and it attaches by base pairing to restriction fragment of complementary sequence

47. 5) Autoradiography
Rinse away unattached probe
Autoradiograph showing hybrid DNA fragment

49. Ultracentrifugation
Equilibrium density gradient ultracentrifugation in CsCl is one of the most commonly used DNA separation procedures.
At high speeds, a linear gradient of CsCl is established.

50. Mixture of DNA, RNA and protein migrating through this gradient separate into discrete bands at position where their densities are equal to density of CsCl.
DNA mol. with high Guanine and Cytosine content are more dense than those with a higher proportion of adenine and thyamine.
The difference helps separate heterogenous mixtures of DNA fragments
Single stranded DNA denser than the double stranded DNA, so the two can be separated by equilibrium density gradient ultracentrifugation.