QUIZ 2

A probe is a labeles, single-stranded DNA or RNA sequence used to detect complementary sequences through base pairing .

-Detecting specific DNA/RNA sequences, diagnosing genetic disoders, studying gene expression, identifying pathogens, mapping genes.

-Radioactive labeling (^32P, ^35S) - sensitive, but hazardous) and Non-radioactive labeling (biotin, digoxigenin, flurescent dyes - safer, stable, versatile)

Biotin is attached to the probe; detection uses streptavidin or avidin conjugated to an enzyme or flurophore.

DIG is incorporated into the probe, detection uses anti-DIG antibodies linked to an enzyme or fluorophore.

Techniques that detect specific DNA or RNA sequences by allowing labeled probes to bind to complementary nucleic acids.

Southern blot → DNA
Northern blot → RNA
In situ hybridization (ISH) → nucleic acids in tissues/cells
Microarrays → many sequences at once

Target nucleic acid is immobilized on a surface (e.g., blot membrane), and a labeled probe binds to it.

Probes are immobilized on a surface, and the target nucleic acid (often fluorescently labeled) binds to them.

A biotinylated end (can be 5′ or 3′ biotin-labeled).

By carefully controlling temperature, salt concentration, and probe design (length, GC content).

Safer, longer shelf-life, and allows multiple detection methods.

Often less sensitive than radioactive labeling.

They allow detection of mutations, infectious agents, and gene expression changes.

PCR (Polymerase Chain Reaction) is a cell-free method for amplifying DNA, Allows parallel amplification of DNA sequences from multiple samples.

Because of its simplicity, speed, and versatility, PCR allows a single person to rapidly amplify specific DNA regions (e.g., all exons of a gene from many patients), which was previously labor-intensive.

DNA template (complex DNA such as genomic DNA)
Two single-stranded oligonucleotide primers (20+ nucleotides, specific, complementary)
Heat-stable DNA polymerase (e.g., Taq)
dNTPs (nucleotides)
Buffer with Mg²⁺
Thermal cycler (for temperature changes)

Denaturation (≈95°C): DNA strands separate.
Annealing (≈50–65°C): Primers bind to complementary target sites.
Extension/elongation (≈72°C): DNA polymerase synthesizes new DNA strand from primers.

Primers are designed to perfectly base-pair with their target sequence (≈20 nucleotides long). Their binding strength depends on length, GC content, and base-pair complementarity.

One primer binds each strand at opposite ends of the target sequence, flanking the DNA of interest. This ensures amplification occurs in both directions and yields a defined-length product.

Initially, products have variable ends, but after ~2 cycles, precise-length products appear (defined by primer sites). These accumulate exponentially, resulting in millions of copies of the target DNA.

The DNA product generated by PCR, representing the amplified target sequence.

By first converting RNA into complementary DNA (cDNA) using reverse transcriptase. This is called RT-PCR (Reverse Transcription PCR).

Routine PCR: Only detects presence/absence or sufficient product.
qPCR: Measures the amount of DNA produced, allowing relative or absolute quantification.

A specialized form of qPCR where amplification is monitored during the reaction using fluorescent reporter molecules. It allows quantification in real-time, not just at the endpoint.

During the exponential phase (≈16–25 cycles), the amount of PCR product is directly proportional to the amount of starting DNA, making it ideal for quantification.

Extremely sensitive (can amplify from tiny or degraded samples, even single cells)
Fast and simple
Robust (works on preserved or formalin-fixed tissue)
Versatile (used in diagnostics, research, forensics, archaeology)

Scaling up to very large DNA quantities is not practical.
PCR usually amplifies fragments <10 kb, while cloning can handle hundreds of kb to >1 Mb.

PCR requires repeated heating (denaturation at ~95°C). Heat-stable polymerases (from thermophiles in hot springs/vents) survive these cycles.

Pfu (from Pyrococcus), Vent™, and Deep Vent™ polymerases. These have 3′→5′ exonuclease proofreading activity and lower error rates.

ag phase: Few products, not detectable.
Exponential phase (cycles ~16–25): Product doubles each cycle, proportional to input DNA (used for quantitation).
Stationary/saturation phase: Reaction slows, efficiency decreases as primers and reagents are depleted.

Reactants (primers, nucleotides) become limiting, and accumulated products compete for primer binding.

Alternative approaches that amplify DNA at constant temperature (instead of cycling), used in some modern diagnostic assays.

A short DNA or RNA sequence that is complementary to a target sequence and used to detect that target through hybridization.

Because hybridization alone isn’t visible; a label/tag is required to detect or visualize the probe after binding.

Colorimetric, enzymatic, fluorescent, and chemiluminescent labels.

DIG is a plant-derived steroid used as a safe, non-radioactive label. It is detected using high-affinity anti-DIG antibodies linked to fluorescence, enzymes, or chemiluminescent reporters.

Through anti-DIG antibodies, which can be conjugated to:
Fluorescent dyes (glow under light)
Enzymes (cause color change)
Chemiluminescent molecules (emit light in chemical reactions)

Stringency is how strictly a probe binds only to perfectly complementary sequences. High stringency = very specific, low stringency = more tolerant of mismatches.

Raise the hybridization temperature → mismatched base pairs become unstable and dissociate.

Lower the hybridization temperature → imperfectly matched sequences can still bind.

Ramp speed is how quickly temperature changes (cooling/heating). Rapid cooling prevents DNA from finding perfect complements (partial mismatches form), while slow cooling allows correct complementary strands to reanneal.

DNA strands fail to find perfect matches, leading to unstable partial pairings or single-stranded DNA.

Strands have time to find correct complementary partners, forming stable double-stranded DNA.

A short nucleic acid sequence (<100 nucleotides) often used as primers or probes.

The annealing temperature in PCR sets stringency:
Too low → primers may bind nonspecifically.
Too high → primers may fail to bind.
Optimal temperature = specific primer binding to the target sequence.

At high temperature, only perfectly complementary sequences have enough hydrogen bonds to remain paired; mismatches dissociate.

At low temperature, probes/primers can stick to partially complementary sequences since weak hydrogen bonding is enough to hold them together.

Temporary unwinding/separation of DNA bases, especially at the ends, even at room temperature.

Higher temperature increases kinetic energy, destabilizes hydrogen bonds, and favors DNA staying single-stranded.

They stabilize DNA ends and protect single-stranded regions from degradation.

High temp → high stringency, only perfect matches bind.
Low temp → low stringency, mismatches can bind, risking non-specific products.

Unlabeled sample DNA/RNA is immobilized; a labeled probe binds to complementary sequences.

Probes are immobilized; labeled sample DNA/RNA binds and produces the detectable signal.

Reverse hybridization, where many probes are immobilized and the sample is fluorescently labeled.

Detect specific DNA sequences in large genomes, confirm recombination events, and analyze DNA fragment sizes.

Digest genomic DNA with restriction enzymes.

Southern = DNA is immobilized, Northern = RNA is immobilized.

It can analyze large DNA fragments, provide copy number info from band intensity, and confirm correct genomic integration.

The relative abundance or copy number of DNA in the genome.

Standard: probe is labeled.
Reverse: sample is labeled.

Hybridization can detect large DNA fragments (e.g., 13 kb), which PCR cannot efficiently amplify.

A chip (usually glass or silicate) with thousands of immobilized DNA probes used to study DNA, RNA, or cDNA hybridization.

Pre-determined DNA oligonucleotides (probes), each spot containing many copies of the same sequence.

The sample DNA/RNA/cDNA is fluorescently labeled, not the probe.

By measuring fluorescence intensity at each probe spot; brighter spots = more hybridization.

Because the signal is too weak; many hybridization events per spot are needed for detectable fluorescence.

Ensuring stringency—all probes must function under the same buffer and temperature conditions.

The relative abundance of the complementary sequence in the sample.

Label each sample with different fluorescent dyes (e.g., red & green) and hybridize both to the same array.

Equal expression in both samples (both red and green fluorophores present).

No hybridization occurred → no expression in either sample.

cDNA is more stable than RNA, making hybridization more reliable.

Detecting single nucleotide polymorphisms by testing for mismatches or by single nucleotide extension with labeled nucleotides.

DNA hybridizes to a probe, then DNA polymerase adds a fluorescently labeled nucleotide at the SNP site. The fluorescence color reveals the SNP identity.

Classic: Detect mismatches by hybridization signal strength.
Modern: Use single nucleotide extension with fluorescently labeled nucleotides.

They allow high-throughput, genome-wide analysis in a single experiment.

The longer the DNA and the more cycles of replication, the greater the chance that DNA polymerase introduces errors. Mutations accumulate over repeated amplification.

Because the amplified gene will be expressed as a protein. Mutations could alter protein structure/function, so high-fidelity polymerases are used and the sequence must be verified afterward.

DNA template (dsDNA)
Primers (forward + reverse)
DNA polymerase (heat-stable, e.g., Taq)
dNTPs (building blocks)
Buffer, salts, Mg²⁺
Thermocycler (to rapidly change temperatures)

Denaturation (~95°C): dsDNA separates into single strands
Annealing (~55–65°C): primers bind to complementary target sequence
Extension (~72°C): DNA polymerase extends primers, synthesizing new DNA

After cycle 2, and it then grows exponentially with each cycle.

It is heat-stable, surviving repeated cycles of near-boiling denaturation temperatures.

Polymerase degradation, depletion of dNTPs or primers, and accumulation of inhibitors cause plateau phase (no further increase in product).

Lag phase: few detectable products yet
Exponential/log phase: product doubles each cycle
Plateau phase: reaction slows/stops due to resource limits

By adding fluorescent dyes that bind dsDNA (e.g., SYBR Green). Fluorescence increases as more product forms.

qPCR uses a probe with a fluorophore + quencher. The probe fluoresces only when degraded by DNA polymerase during extension, ensuring signal comes only from the correct target.

The cycle threshold (Ct value) — the cycle at which fluorescence crosses a set threshold — correlates with the starting amount of DNA template. Fewer cycles = more template initially present.

RNA is reverse transcribed into cDNA, then amplified and quantified by qPCR → used to measure gene expression.

They are in vast excess and much smaller, allowing them to rapidly anneal before templates re-hybridize.

Different probes with different fluorophores can detect multiple targets in the same reaction tube.