MIDTERM LEC: ANALYSIS & CHARACTERIZATION OF NUCLEIC ACIDS & PROTEINS Flashcards
Set of techniques & methods used to study & understand the structure, function, interactions, & characteristics of nucleic acids & proteins in biological systems
NUCLEIC ACID/PROTEIN ANALYSIS
what are the methods for NUCLEIC ACID/PROTEIN ANALYSIS
A. Restriction Enzyme Mapping of DNA
B. CRISPR Enzyme Systems
C. Hybridization Technologies
• Involves restriction enzymes (endonucleases): type II is commonly used, with 4-6 bp recognition/restriction sites, or binding/cutting sites on DNA
▪ Used to identify the position of restriction sites in a DNA fragment in relation to each other
▪ Developed using small circular bacterial plasmids
RESTRICTION ENZYME MAPPING OF DNA
RESTRICTION ENZYME MAPPING OF DNA what type is commonly used???
type 2
Developed using small circular bacterial plasmids
RESTRICTION ENZYME MAPPING OF DNA
Restriction enzyme digestion of DNA fragment/region = pattern of fragments
Used to identify that DNA
▪ Resulting differences in the size/number of restriction fragments
▪ Useful in epidemiological studies
▪ The basis of the 1st molecular-based
human ID & mapping methods
▪ Clinical analysis of structural changes in chromosomes associated with disease
Restriction fragment length
polymorphisms (RFLPs)
RESTRICTION MAPPING USES/APPLICATIONS??
▪ Gives an idea of genome sequence
▪ Used to map genomes when sequence information is unknown
▪ Can be used to introduce primers for sequencing
▪ Used for ID & characterization of naturally-occurring plasmids & to engineer the construction of recombinant plasmids
▪ Found in archaea & bacteria
▪ Guides a common enzyme to specific sites determined by RNA components
▪ Useful for manipulation of both DNA sequence & RNA expression
CRISPR ENZYME SYSTEMS
CRISPRs MEANING?
CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS
▪ Protective system using the invading DNA to target itself
▪ Encodes an endonuclease: CRISPR-associated protein (Cas)
CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPRs)
CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPRs) Encodes an endonuclease:
CRISPR-associated protein (Cas)
CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS
Types:
Type 1 and 2 targets???
dsDNA
CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS
Types:
Type 3 targets??
ssDNA & RNA
Hybridization reaction that is used to analyze the nucleic acid content of an unknown sample
HYBRIDIZATION TECHNOLOGIES
formation of H bonds between 2 complementary strands of nucleic acids
Hybridization
In hybridization technologies: 1 strand is unlabeled & 1 strand is labeled (with isotopes/ fluorescence) = _____
probes
▪ ss fragment of nucleic acid(NA)/protein with a detectable signal that specifically binds to complementary sequences/ target protein
▪ Purpose: identify 1 or more sequences of interest within a large amount of NA
PROBES
southern blot target???
DNA
northern blot target???
RNA
Western blot target??
Protein
south western blot target???
protein
eastern blot target???
protein
far eastern blot target???
lipids
southern blot probe???
nucleic acid
Northern blot probe???
nucleic acid
western blot probe??
protein
South western blot probe???
DNA
eastern blot probe??
Protein
far easter blot probe??
none
target NA has to contain the sequence of interest
In order the probe to bind
Denaturation of dsDNA
probes before use:
❖ Heating the probe (95ºC, 10-15 min) in hybridization solution
❖ Treating with 50% formamide/2x SSC at a lower temperature for a shorter time (75ºC, 5-6 min)
▪ Early methods: Fragment of gene was cloned on a bacterial plasmid 🡪 isolated by restriction enzyme digestion & gel purification 🡪 labeling & denaturation of fragment 🡪 applied to Southern/Northern blot
▪Isolation of sequence of interest from viral genomes
In vitro organic synthesis of a predetermined sequence 🡪 only for short, oligometric probes
▪ Synthesized using PCR
DNA PROBE
RNA PROBES
Source: transcription from a synthetic DNA template in vitro
▪ Predesigned systems commercially available
▪ Southern blot: coding strand transcript
▪ Northern blot: antisense transcript ▪Labeled with a radioactive/modified
nucleotide for production of signal
RNA PROBE
OTHER NA PROBE TYPES
Synthetic & resistant to nucleases:
peptide NA probes, locked NA probes, unlocked NA probes
products of a generalized response to a specific antigen
Polyclonal antibodies
