MOLECULAR BIOLOGY

LABORATORY TECHNIQUES

Nucleic Acids
Eugene John F. Balmores Jr., MD
OUTLINE
I. Nucleic Acid Extraction
II. Nucleic Acid Detection
Hybridization, PCR
III. Nucleic Acid Sequencing
IV. Other Analyses
RFLP, Microarrays
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
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
 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.
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.
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.
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.
 NUCLEIC ACID PURIFICATION
1. SALT PRECIPITATION METHOD 2. USING ORGANIC SOLVENTS

• https://youtu.be/TbxAli7WVP4 • https://youtu.be/Vz2uu5iyT0I

3. USING SILICA COLUMNS 4. USING MAGNETIC BEADS

• https://youtu.be/ZN1bZ6Q_mG4 • https://youtu.be/SJ6c40lJuK0
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
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
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
SOUTHERN BLOTTING
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
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
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
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
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
• https://youtu.be/mZA3fdKijHY
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=jMLIaxxY6-8
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
PRACTICE
EXERCISES
THANK YOU