nucleic acids

• 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

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).

• 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.

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

• 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.

• 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

• 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 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

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

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

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

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

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

• Solid Phase Assays - in a solid support medium • Solution - based assays • In situ hybridization assays (on a slide)

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

• 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

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.

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

• 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

• 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

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

• 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

• 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

• 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.