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
Southern Blot
- DNA extracted →fragments
- Electrophoresis - separates fragments
3 & 4. Transfer or blot DNA fragments from gel
to membrane - Add labelled probe
- Bound DNA shows up on film
Solution-Based Assays
Similar to Solid Phase
• Probe (labeled) and target are mixed
• Hybrids are formed
• Separate to remove unbound, labeled probes
• Read amount of bound label
• Amount of bound label is proportional to target DNA present.
In-Situ Hybridization Assays
Performed on cells, tissues or chromosomes fixed to a microscope
slide
• DNA is heat denatured
• A labeled probe is added
• Will hybridize with target sequence as slide cools
Good morphologic context of where target DNA is located
Colorimetry/ fluorescence used - if probe is labelled with an enzyme
Restriction Fragment Length Polymorphism (RFLP)
- Evaluates differences in DNA sequences
- Genomic DNA is extracted from a sample and purified and quantitated.
• A restriction enzyme (endonuclease) cleaves target DNA at a specific site
• Mutations or changes in the DNA sequence may cause the DNA to cleave
differently - get different DNA fragment length
• Measured using Southern Blot to identify the different lengths of the DNA
fragments.
• PCR can be used to amplify the target sequence prior to RFLP
Used in:
• Forensic/ paternity cases -to establish identity or non-identity
• Also used to ID a gene associated with a disease
Amplification Techniques
• Often the amount of target DNA is in such a low concentration that it
needs to be increased (amplified) to be detected.
• Used to ↑ sensitivity of hybridization assays to permit detection
(otherwise may get false negative)
• May be achieved by:
- Target amplification
- Probe amplification
- Signal Amplification
Target Amplification
Involves:
• In vitro methods for enzymatic replication of a target molecule (target DNA
sequence) to levels at which it can be readily detected
• Allows target sequence to be identified & further characterized
- Using southern blot or another technique
- Amplified target DNA sequences = amplicons (copies of the original DNA sequence)
- Polymerase chain reaction (PCR): most widely used
Polymerase Chain Reaction (PCR)
- Millions of copies of target DNA produced
- Greater analytical sensitivity
- Procedure:
- Target DNA denatured by heating - Reagents added; primer (AKA probe) binds to target DNA sequence - Taq Polymerase extends the primers - Denatured again - Process repeated 20-30x
- Amplicons (end product) can be analyzed by gel electrophoresis, Southern blot etc.
- Latest PCR innovation = Real time RT-PCR: Direct measurement of amplicon in exponential phase
PCR Limitations
• Expense
• Special equipment (thermocyclers)
• Sample contamination (potential aerosol contamination from one
sample to another)
- Must work in a sterile, clean environment
• Non-specific annealing
• Degree of stringency (stability of bonding between target DNA & probe
ie. primer)
Other techniques evolved to overcome some of the problems
Probe Amplification
• Several techniques amplify the detection molecule or probe itself,
instead of target.
• Ligase chain reaction (LCR)
- Uses 2 pairs of labeled probes that are complementary for 2 short-target
DNA sequences in close proximity
- After hybridization, DNA ligase interprets break between ends as a nick
& links probe pairs.
Signal Amplification
• Techniques that increase signal strength by increasing the concentration of
the label
- Can use multiple labels to a probe
- Multiple short probes per target (several sandwich hybridizations)
• 2-Tiered probe system
- Part of primary probe attaches to target - Part of primary attaches to 2nd probe (Labeled)
• Branched DNA assay (bDNA)
- Similar to above
- Tertiary probe labelled (attaches to 2ndary probe)
- Assays have been developed for Hepatitis B virus, HCV, HIV-1 and
cytomegalovirus
Branched Chain Amplification
Several different probes are used to amplify the signal rather than the target DNA itself. The first probe captures the target. Amplifer 1 binds in a different place and forms a base for amplifer 2, the branched chain. Amplifier 3 contains the signal.C
Applications of Molecular Assays
• Detect infectious organisms
• Detect gene rearrangements & chromosomal translocations & breakage (eg.
Philadelphia chromosome in CML)
• Detect changes in oncogenes & tumor suppressor factors
• Aid in prenatal diagnosis of an inherited disease or carrier status (eg. Sickle
Cell Anemia, Cystic Fibrosis, Von Willebrand Disease)
• Identify polymorphic markers used to establish identity/nonidentity (eg.
Paternity cases, forensics)
• Aid in donor selection (transplant compatibility)
Summary
- Nucleic acid probes can detect DNA/ RNA in human, bacterial or viral genomes
- Unamplified are generally qualitative
- Southern Blot
- Northern Blot
- In Situ Hybridization
- Restriction Fragment Length Polymorphism (RFLP)
• Amplified (quantitative) more sensitive
- Target Amplification (ex. PCR) - Probe Amplification (Ligand Chain Reaction) - Signal Amplification (Branched DNA Assay)
