2. OUTLINE
I. Nucleic Acid Extraction
II. Nucleic Acid Detection
Hybridization, PCR
III. Nucleic Acid Sequencing
IV. Other Analyses
RFLP, Microarrays
3. I. NUCLEIC ACID EXTRACTION
- Plays a vital role in molecular biology as the
primary step for many downstream
applications
- Many modifications have been introduced to
the original 1869 method
- Can be roughly divided into:
- Cell disruption/lysis
- Nucleic Acid Purification
4. CELL LYSIS
• Main aim is to disrupt the cell wall and/or
membranes
• Lytic enzymes, chaotropic agents, detergents
• Grinding, shearing, bead beating, shocking
• https://youtu.be/BpXZBq3uQPA
5. NUCLEIC ACID PURIFICATION
1. SALT PRECIPITATION METHOD
• After lysis with detergents that
solubilize cell components and inhibit
nucleases, proteins are denatured and
eliminated by salt precipitation; DNA is
then precipitated with isopropanol,
washed with ethanol and resuspended
in a buffer.
2. USING ORGANIC SOLVENTS
• Addition of solvents leads to an upper
aqueous phase (containing NAs) and
an organic phase containing proteins
solubilized in phenol and lipids
dissolved in chloroform.
3. USING SILICA COLUMNS
• In the presence of high concentrations
of chaotropic salts, nucleic acids are
adsorbed on a silica column; with low
ionic strength buffer at neutral or
slightly alkaline pH, NA is eluted out of
the column.
4. USING MAGNETIC BEADS
• Magnetic beads have polymers that
have a high affinity to NAs. As
magnetic field is placed on the wall of
the tube with cell lysate, NAs on the
bids are separated from the dissolved
contaminants.
6. NUCLEIC ACID PURIFICATION
1. SALT PRECIPITATION METHOD
• https://youtu.be/TbxAli7WVP4
2. USING ORGANIC SOLVENTS
• https://youtu.be/Vz2uu5iyT0I
3. USING SILICA COLUMNS
• https://youtu.be/ZN1bZ6Q_mG4
4. USING MAGNETIC BEADS
• https://youtu.be/SJ6c40lJuK0
7. QUALITY OF EXTRACTED NA
• Concentration
• Absorbance at OD = 260nm
• 1A = 50ng/uL
• https://youtu.be/BpXZBq3uQPA
• Purity
• A260 / A280
• Integrity
• Electrophoresis in a 0.7% agarose gel
• https://youtu.be/awRRnEPtSic
• Functionality
• Application in PCR
8. II. NUCLEIC ACID DETECTION
- Detecting NAs are important for a wide variety
of studies, including the mapping of genes to
chromosomes and the analysis of gene
expression
9. NUCLEIC ACID HYBRIDIZATION
• At high temperatures (e.g., 90
to 100°C), the complementary
strands denature, yielding
single-stranded molecules. If
such denatured DNA strands
are then incubated under
appropriate conditions (e.g.,
65°), they will renature to
form double-stranded
molecules as dictated by
complementary base pairing
11. NUCLEIC ACID HYBRIDIZATION
• Nucleic acid hybridization can be used to detect
homologous DNA or RNA sequences not only in
cell extracts, but also in chromosomes or intact
cells—a procedure called in situ hybridization.
• https://youtu.be/QI7A9XM1vuE
• https://youtu.be/eMkY2jzgWjg
12. POLYMERASE CHAIN REACTION
• Much more sensitive technique for
detecting cellular DNA or RNA sequences
than is Southern or Northern blotting
• https://youtu.be/db0HzFTJtCs
• https://youtu.be/9pPg9-dw33w
• https://youtu.be/Vd38iS_W7ww
13. III. NUCLEIC ACID SEQUENCING
- Is an analytical procedure of decoding
the DNA sequence to determine the
order of the base pairs of the
nucleotides present in a stretch of DNA
- Central dogma of molecular biology
- DNA can undergo self-replication to form another
DNA as well as undergo transcription to form
RNA. The order of the nucleotides is essential for
the formation of the RNA and its further
translation to the coded protein leading to the
translation of the genetic information into the
structural proteins
14. SANGER SEQUENCING
• Developed by Frederick Sanger enabled the
sequencing of bacteriophage phi X 174 which
contains approximately 5375 nucleotides thus
becoming the first fully sequenced genome in the
year 1977
• In 2003, the Human an international consortium
effort, successfully sequenced and mapped the
entire human genome, which came to an end after
13 years of research around many laboratories in
the world
• https://youtu.be/e2G5zx-OJIw
15. NEXT GENERATION SEQUENCING
(NGS)
• Launched by Roche’s 454 technologies in
2005.
• The key feature NGS is a parallel
sequencing process producing several
thousands of sequences simultaneously.
• These high-throughput sequencers reduced
the cost of DNA sequencing. This is
achieved by miniaturization of sequencing
reactions
16. IV. OTHER NA ANALYSES
- Restriction Fragment Length Polymorphism
- is a difference in homologous DNA
sequences that can be detected by the
presence of fragments of different
lengths after digestion of the DNA
samples in question with specific
restriction endonucleases
- https://www.youtube.com/watch?v=jMLIa
xxY6-8
17. IV. OTHER NA ANALYSES
- Microarray
- is a hybridization of a nucleic acid
sample (target) to a very large set of
oligonucleotide probes, which are
attached to a solid support, to determine
sequence or to detect variations in a
gene sequence or expression or for gene
mapping
- https://youtu.be/6ZzFihESjp0
