Analysis and Characterization of Nucleic Acids and Proteins
Detection of Nucleic Acids and Proteins
● The advent of molecular cloning has enabled the isolation and characterization of
individual genes from eukaryotic cells (multicellular).
● Understanding the role of genes within cells, however, requires analysis of the
intracellular organization and expression of individual genes and their encoded
proteins.
● These approaches are important for a wide variety of studies, including the mapping of
genes to chromosomes, the analysis of gene expression, and the localization of
proteins to subcellular organelles.
● The same general procedures are also used to isolate specific genes as molecular
clones.
Gene Mapping (Genome Mapping)
● Refers to the process of determining the location of genes on chromosomes.
○ to identify the location of gene in chromosome and its distances with each
other
○ Uses computer-based programs, to identify easily sequence (DNA template)
Nucleic Acid Hybridization
● The key to detection of specific nucleic acid sequences is base pairing between
complementary strands of RNA or DNA.
○ to detect and identify specific DNA or RNA sequences
○ in real life scenario it is used to identify specific genes, pathogens (bacteria
viruses, parasite)
○ uses to know the what is happening in genetic mutation)
● At high temperatures like 90 – 100 degrees Celsius, the complementary strands of
DNA separate or denature, yielding single-stranded molecules.
● If such denatured DNA strands are then incubated under appropriate conditions like
65 degrees Celsius, they will renature to form double-stranded molecules as dictated
by complementary base pairing—a process called nucleic acid hybridization.
○ Denaturation(95*C celsius)- break the quality of molecules For example there
is a DNA, this will be exposed in denaturation, hydrogen bonding will be
removed that connects the two base pairs in a nucleotide, the double stranded
will become single stranded
○ Renaturation (65*C)- introduces DNA probe, reagent
radiolabeled/radioactive, is a single stranded will be exposed in a single strand
DNA to become a radiolabeled
○ this process will be used to identify pathogens
Fluorescence in Situ Hybridization (FISH)
● a molecular cytogenetic technique that uses fluorescent probes to identify and
visualize specific DNA sequences or entire chromosomes within cells, aiding in the
diagnosis of genetic disorders and cancer
○ incorporated fluorescent dye
○ can be viewed with fluorescent microscope (special)
○ tested 1 chromosome (many DNA)
○ will be going to undergo denaturation and renaturation
● the genes that fluorescence will be the one that is determining
Western Blot
- confirmatory for HIV
- Screening: ELISA (Enzyme-linked Immunosorbent Assay)
- uses test kit (single line- neg; double line- positive, single line in control
only- invalid)
- if tested positive, will be transfer to San Lazaro Hospital (reference
Lab for HIV)
- if positive, legit positive
- check for proteins/ antibodies
Southern Blot
● checks for DNA
● discovered by Edwin Southern
● allow to visualize/get specific DNA that want to be obtained
● Specifically, purified DNA from a biological sample, such as blood or tissue, is digested
with a restriction enzymes, and the resulting DNA fragments are separated by using
an electric current to move them through a sieve-like gel or matrix, which allows
smaller fragments move faster than larger fragments.
● The DNA fragments are transferred out of the gel or matrix onto a solid membrane,
which is then exposed to a DNA probe labeled with a radioactive, fluorescent or
chemical tag.
● The DNA to be analyzed is digested with a restriction endonuclease, and the digested
DNA fragments are separated by gel electrophoresis.
○ For example in a cell, inside the nucleus there is DNA, the main goal is to get
the DNA from the nucleus, to get that cell wall and nuclear membrane should
be destroyed by using detergent, once DNA exits the nucleus, labeled it as
Gene A.
○ Before you get a specific DNA, this should be introduced with a restriction
enzyme (degrade the whole DNA molecule to form many DNA fragments), the
whole process is called Restriction Enzyme Digestion.
○ Gel electrophoresis will be performed using gel (agarose, polyacrylamide), in
which each fragments will be run in cathode and let the bands separate.
○ Then will be adding DNA probe, but gel is fragile so filter paper will be used
and will be submerged with DNA probe, the filter paper will then be put on the
top of the gel and let absorption happen to copy bands, DNA probe will bind to
specific DNA segments
○ To visualize, use X-ray film. will be visualized because of dye (fluorescence)
Northern Blot
● checks for RNA; same with southern blot but DNA
● by Edwin Southern
● a variation of the Southern blotting technique, hence its name, that is used for
detection of RNA instead of DNA.
● In this method, total cellular RNAs are extracted and fractionated according to size by
gel electrophoresis.
● As in Southern blotting, the RNAs are transferred to a filter and detected by
hybridization with a radioactive probe.
Detection of Small Amounts of DNA or RNA br PCR
Polymerase Chain Reaction
● Amplification of DNA by the polymerase chain reaction is a much more sensitive
technique for detecting cellular DNA or RNA sequences than is Southern or Northern
blotting.
○ amplify- to make many copies of molecules
○ just need 1 copy of DNA unlike blot that needs 100 copies or more to proceed
with the test
● Approximately 100,000 copies of a DNA or RNA sequence are required for detection
by blot hybridization.
● In contrast, PCR can amplify single copies of DNA or RNA after reverse transcription
to readily detectable levels.
● Applications:
○ COVID
○ Forensic (crime scene)
○ Agriculture (breeding of plants)
○ Medicine (to diagnose and identify microorganisms)
● Steps:
○ Denaturation- 95*C
○ Annealing- 55*C- 65*C
○ Extension- 72*C
Materials needed for PCR
Taq polymerase
● a DNA polymerase that's used to amplify DNA in the polymerase chain reaction (PCR)
○ heat resistant
○ thermophilic
○ came from bacteria Thermus aquaticus (bacilli)- found in hot spring/geysers
Primers
● are short, single-stranded DNA sequences that flank the target region to be amplified,
providing a starting point for DNA polymerase to synthesize new DNA strands
○ needed by Taq polymerase to start
○ in a short sequence of nucleotides; provides starting point in DNA synthesis
DNA Template
● a single-stranded DNA sequence used to synthesize a new DNA, RNA, or protein
polymer during processes like replication, transcription, and translation
○ the one that is amplify in PCR
Nucleotides
● a compound consisting of a nucleoside linked to a phosphate group. Nucleotides form
the basic structural unit of nucleic acids such as DNA.
○ glucose backbone- where nucleotides are attached and connected
● building blocks of DNA for synthesis
Process of PCR
Denaturation (95*C) ● There is one DNA molecule, denaturation wll
destroyed hydrogen bonding, base pairs will be
separated from each other to form 2 single stranded
DNA
Annealing (55-65*C) -came from metal industry, where iron will be exposed in a
high temperature then low temperature to destroy structure
and have internal defects
● 2 primers will attached to the 2 single stranded from
denaturation process, primers have complementary
nucleotides that is complementary to strands
● after connecting, taq polymerase will act, when it
binds, extension process will start
Extension (72*C) ● increases temperature, due to Taq polymerase, it
will extend the strands until the end of strand
● the whole process will form 2 double strand and so
on from 1 double strand
Antibodies as Probes for Proteins
Proteins
● Studies of gene expression and function require the detection not only of DNA and
RNA, but also of specific proteins.
○ large biomolecules, function as enzymes, immunoglobulins, keratin, collagen,
hemoglobin
Antibodies
● It takes the place of nucleic acid probes as reagents that can selectively react with
unique protein molecules.
○ protective proteins against antigens, and infection
● Are proteins produced by cells of the immune system (B lymphocytes) that react
against molecules (antigens) that the host organism recognizes as foreign
substances—for example, the protein coat of a virus.
● The immune systems of vertebrates are capable of producing millions of different
antibodies, each of which specifically recognizes a unique antigen, which may be a
protein, a carbohydrate, or a non biological molecule.
Immunoprecipitation
● Antibodies are used to isolate the proteins against which they are directed.
● Typically, cells are incubated with radioactive amino acids to label their proteins.
● Such a radiolabeled cell extract is then incubated with an antibody, which binds to its
antigenic target protein.
○ method to isolate specific antigen from a mixture
○ uses antigen-antibody reaction that will be read in a Western Blot
