The document provides an overview of nucleic acid blotting techniques, including Southern, Northern, Western, colony, and dot blotting, highlighting their roles in the immobilization and detection of nucleic acids. It also discusses various DNA sequencing methods such as Sanger's chain termination method, Maxam-Gilbert chemical sequencing, and next-generation sequencing techniques, along with their principles and applications. Key processes like sample preparation, sequencing methods, and detection mechanisms are summarized, emphasizing advancements in sequencing efficiency and accuracy.

1. R.T.VINODHINI
UNIT-5

2. NUCLEIC ACID BLOTTING TECHNIQUE
• Blotting - immobilization of sample nucleic acid in solid support.
• Blotted nucleic acids - target in hybridization
• Specific detection

3. TYPES
• Southern blotting
• Northern blotting
• Westernblotting
• Colony blotting
• Dot blotting

4. SOUTHERN BLOTTING

5. NORTHERN
BLOTTING

6. WESTERN BLOTTING

7. DOT BLOTTING
• Sample (DNA/RNA) - fragmented on to a nitrocellulose filter in the form of
dots.
• DNA - denatured & the filter is baked at 80ºc to (fix)
• Filter - pre treated to prevent non-specific binding of the probe to the filter.
• Filter - treated with appropriate radioactive single stranded DNA probe
• Washed - remove free probe.
• The hybridized probes are detected by autoradiography.

9. COLONY BLOTTING

11. DNA SEQUENCING
• Determining order of nucleotides (A,T,
G &C)
• Methods:
• Sanger -ChainTermination Sequencing
method
• Maxam andGilbert -Chemical
Sequencing method
• Next generation sequencing

12. SANGER METHOD
• Chain termination sequencing/dideoxy method
• PCR based method
• Modified DNA replication reaction
• Growing chains are terminated by dideoxynucleotides

13. SANGER METHOD
• Denaturing DNA – Single stranded DNA template
• DNA primer
• DNA Polymerase
• Normal deoxynucleosidetriphosphates (dNTPs) and modified
dideoxynucleosidetriphosphates (ddNTPs)
• ddNTPs –Terminate DNA strand elongation – lack 3’OH
• 3’OH – necessary for phosphodiester bond formation
• Cease extension of DNA
• Labelling - radioactively or fluorescently labeled for detection in automated
sequencing machines

17. MAXAM GILBERT METHOD
• Nucleobase - chemical modification of DNA (radioactive labelling of 5’ end
of DNA)
• Modification &Cleavage at specific sites
• Purines (A+G) are depurinated - formic acid
• Guanines (and to some extent the adenines) - methylated by dimethyl
sulfate
• Pyrimidines (C+T) - hydrolysed using hydrazine
• Cytosine – Hydrazine + NaCl
• Cleavage ofSugar Phosphate backbone - Piperidine

20. NEXT GENERATION SEQUENCING
• Sequence DNA and RNA - quickly and cheaply than conventional
sequencing.
• 454 Pyrosequencing
• Ion torrent semiconductor sequencing
• Reversible terminator sequencing (Illumina)
• 3′-O-blocked reversible terminators
• 3′-unblocked reversible terminators

21. 454 PYROSEQUENCING
• Sequencing by synthesis
• Complementary strand
• Four different dNTPs are then sequentially made to flow in and out
• Correct dNTP - release of pyrophosphate
• Pyrophosphate is converted intoATP
• Luciferase-catalysed conversion of luciferin to oxyluciferin

23. ION TORRENT SEMICONDUCTOR
SEQUENCING

24. ILLUMINA SEQUENCING
• Reversible terminator
sequencing
• Modified nucleotides - 3’-O-
azidomethyl group
• Nucleotides – labelled with
different fluorophores

25. 3’-O-BLOCKED REVERSIBLE TERMINATORS

26. 3’-O-UNBLOCKED REVERSIBLE TERMINATORS
• Reversible termination group – Linked to
base and fluorescence (reporter &
termination group)
• Different from blocked reversible
terminator:
• 3’-position is not blocked (i.e. the base has
free 3’-OH)
• Fluorophore - same for all four bases
• Each modified base - flowed in sequentially
rather than at the same time