nucleic acids Flashcards
Molecular Techniques
• Assays that target nucleic acid instead of protein.
• Detect changes at the DNA/RNA level instead of protein detected in
immunoassays.
• Basic steps:
• Binding of a nucleic acid to its complementary target nucleic acid
sequence.
• Target nucleic acid may or may not be amplified for
detection/quantitation.
In the clinical lab, molecular techniques are used to identify unique
nucleic acid sequences. This can be done by:
• Enzymatic cleavage of nucleic acids
• Gel electrophoresis
• Enzymatic amplification of target sequences
• Hybridization with nucleic acid probes
Nucleic Acid Chemistry
Deoxyribonucleic Acid (DNA)
• Stores genetic information and directs the synthesis of
specific proteins.
• Comprised of two strands of nucleotides linked by
phosphodiester bonds.
• Each strand is composed of a sugar-phosphate backbone
and attached bases.
• The complementary strands of DNA in the double helix are held
together by hydrogen bonds.
• The complementary base pairs are:
• adenine and thymine (2 H bonds).
• guanine and cytosine (3 H bonds).
Structure of DNA
• A double-helix is the secondary structure of DNA.
• The double-helix is two intertwined polynucleotide chains that run in
opposite directions.(anti-parallel)
• The bases point inward.
Nucleic Acid Chemistry
How ribonucleic acid (RNA) differs from DNA:
- RNA is single-stranded; DNA is double-stranded
- Ribose replaces deoxyribose as the sugar
- Uracil replaces thymine as a purine base
Primary Structure of Nucleic Acids
- Nucleic acids have a sugar-phosphate backbone.
- The 5’ carbon of one nucleotide joined to the 3’ carbon of the next.
- The linkages are phosphodiester bonds.
- The order of the bases along the backbone is unique.
Protein Synthesis
• DNA and RNA work together
• Double-stranded DNA (dsDNA) is enzymatically split into two strands.
• One strand serves as a template for the synthesis of complementary
messenger RNA (mRNA).
- As mRNA is released, the DNA strands reanneal.
* Always follows base pairing rules.
• mRNA attaches to a ribosome and specifies the amino acid to be added to
the peptide chain.
• Codon – a sequence of three DNA or RNA nucleotides that
correspond with a specific amino acid or stop signal during protein
synthesis.
• Transfer RNA (tRNA) brings in the correct amino acid to elongate the protein
chain
The Flow of Genetic Information
• DNA directs synthesis of proteins through replication, transcription and
translation.
• DNA does not direct synthesis of carbohydrates, lipids, NPNs, but enzymes
cause reactions for their synthesis
Nucleic Acid-Based Techniques
• Nucleic acids can be evaluated to detect changes at the DNA or RNA level
before changes in specific protein products would be detectable.
- Nucleic acid hybridization can be used to investigate the following:
* Genetically based disease
* Presence of infectious organisms
* Differences between individuals for forensic and transplant purposes
* Altered cell growth regulation
Nucleic Acid Hybridization
Involves complementary strands of nucleic acid from unrelated sources
binding together to form a hybrid or duplex.
• One strand is of known identity (Probe)
-Short strand of DNA or RNA which is complementary for the base
sequence on the test target; detection molecule.
• One strand is unknown (Test Target)
• New double strand = Hybrid
Molecular Testing
Two major areas:
1. Use of DNA probes to directly detect a specific target
• Solid-phase assays
- Southern blot
- Northern blot
• Solution-based assays
- Protection assays
- Hybrid capture assays
• In situ hybridization assays
- Use of nucleic acid amplification technology to detect a specific target DNA
or RNA
• Nucleic acid amplification
- Polymerase chain reaction (PCR)
- Transcription-mediated amplification (TMA)
- Strand displacement amplification (SDA)
• Probe amplification
- Ligase chain reaction (LCR)
• Signal amplification
- Branched chain DNA assay
Nucleic Acid Probes
Can be:
• Genomic nucleic acids (from pure organisms)
• Cloned DNA (Plasmids - produced in bacteria)
-The base sequence (probe) is inserted into the plasmid vector, put
into the host (bacterium) where it replicates. It can then be
isolated and purified.
• Synthetic DNA (if AA sequence of protein known)
~ Used to detect and quantify nucleic acids in samples
~Evaluation of nucleic acids = earlier detection of cellular changes before
end product produced
Considerations in Nucleic Acid Analysis
Probe must be complimentary for base sequence on test target
Need a means to detect hybridization (ie need a reporter molecule or label):
• Probe is labelled with either:
- Radioactive element – will expose x-ray film where the probe is located
- Biotin - binds to avidin (protein)
- Enzyme - substrate added to produce colorimetric, fluorescent or
chemiluminescent product
Test Target (What we are trying to detect in the sample):
• Nucleic acid must be released from cell
• Nucleic acid must be stabilized to preserve sequence
• If DNA, must be single strand (denatured)
• RNA is naturally single strand
• Does RNA have to be denatured?
-No, it is already single stranded
Stringency
The process of hybridization is controlled by:
- Solution buffer
- Temperature
- Presence or absence of a denaturant
High stringency conditions only accept perfect complementary sequences to base-pair and hybridize. - Low salt concentrations -High temperature - Presence of a denaturant (formamide)
Low stringency conditions the requirement for sequence specificity is
relaxed, and mismatches may be tolerated.
- High salt concentration
- Low temperature
Hybridization Formats
- Solid Phase Assays - in a solid support medium
- Solution - based assays
- In situ hybridization assays (on a slide)
Solid Phase Assays
Southern Blot – Classic Method
• DNA extracted from sample (phenolic reagent)
• DNA digested by restriction enzymes →DNA fragments
• Fragments are separated via gel electrophoresis
• Separated fragments denatured to single strand & transferred to solid
support medium (Nitrocellulose or charged nylon membrane)
• Labelled probe added→ binds to complementary base sequence & appears
as band
- Northern Blot
- Similar to Southern blot; RNA extracted
Memory Aid:
“Down South Right Now”
DNA - Southern Blot
RNA - Northern Blot
