Acid Hybridization Reviewer (HY)

Acid Hybridization Reviewer (HY)

THE ONE PRINCIPLE THAT GOVERNS EVERYTHING

Single-stranded nucleic acids bind to their complementary sequences → forms a hybrid molecule. The probe finds the target. That's it.

MOST TESTED FACTS

Hybrid format is always written as: Target — Probe

• DNA–DNA, DNA–RNA, RNA–RNA

Steps always go: Denaturation (95°C) → Renaturation (~65°C) → Hybridization

vs PCR: Both denature first. PCR 2nd step = annealing via primer. NAH 2nd step = renaturation via probe.

G/C-rich sequences need HIGHER denaturation temp because G≡C has 3 hydrogen bonds vs A=T which only has 2.

Formamide = chemical backup for denaturation (used alongside or instead of heat).

PROBE vs TARGET — KNOW THIS COLD

Probe length rules:

• Longer probe = more specific = better for general detection

• Shorter probe = better for detecting point mutations (addition, deletion, substitution)

• Drawback of short probes = HIGH BACKGROUND NOISE (nonspecific binding)

Target mnemonic — De-Good-Doesn't Blur-A Clear Base Sequence

PROBE TYPES — HIGH YIELD DIFFERENCES

DNA Probe

• Longer = more specific

• Shorter = for mutational analysis but noisy

• Sequence matters — GC content affects temperature needed

RNA Probe — 3 things to memorize

1. Made by in vitro transcription from DNA (plasmid or PCR amplicons)

2. Equal or greater binding affinity than DNA probes

3. More sensitive than DNA probes because it is already single-stranded — no denaturation needed, binds target easily

LABELING — 3 METHODS, 3 LABELS

How to Label (Technique)

End Labeling — label added at the 5' or 3' end using terminal transferase or T4 polynucleotide kinase

Nick Translation — label inserted into strand breaks ("nicks") on DNA; DNA polymerase extends the nick incorporating labeled dNTPs

Random Priming — short probe hybridizes with ssDNA; labeled dNTPs added during extension, generating a new labeled ssDNA (PCR-like process)

What Label to Use

Radioisotopes (32P, 35S, N)

• Detected by autoradiography or X-ray film

• ⚠ NO LONGER FAVORED — hazardous to humans and environment, short half-life means experiments must be done quickly

Non-Radioactive (Biotin or Digoxygenin on UTP/CTP)

• Anti-digoxygenin or streptavidin conjugated to alkaline phosphatase (AP) or horseradish peroxidase (HRP)

• Substrate used: dioxetane or tetrazolium dye

• Detected via chemiluminescence

Fluorescent Dyes (Fluorophores)

• Fluorophore absorbs light at specific wavelength → emits vivid signal

• Quencher molecule suppresses background noise so only probe-bound target fluoresces

• Detected via fluorescence microscope

• This is what FISH uses

One rule applies to ALL labels: Excess/unbound probe must always be washed off to prevent nonspecific binding and false positives.

BLOTTING METHODS — THE BIG TABLE

Common denominator of all blots: They all use probes. They all require blotting. Steps are similar but differ based on target.

SOUTHERN BLOT — STEP BY STEP

Target = DNA | Probe = Nucleic acid | First done by Edwin Southern

Step 1: RE Digestion

• Restriction endonucleases cut at 4–6 bp palindromic sites

• Palindromes: same sequence on both strands when read 5'→3' on each strand

• Common REs: EcoRI, BamHI, HindIII

• RFLP = Restriction Fragment Length Polymorphism — fragment sizes vary between individuals

• Star activity = altered RE specificity under nonstandard conditions

Step 2: Resolution (Gel Electrophoresis)

• Larger fragments migrate slower, smaller fragments migrate faster toward anode

• Smear at bottom = degraded DNA

• Aggregates at top = incomplete digestion

Step 3: Denaturation

• Separates dsDNA → ssDNA for probing

• Use NaOH directly, or HCl for depurination first if fragments are >500 bp

• No heat used here — chemicals only

Step 4: Blotting (Transfer)

Capillary mnemonic — GEL ON TOP! AML: Absorb · Move · Left overnight

Prehybridization step always comes before adding the probe — prevents nonspecific binding by blocking the membrane.

Step 5: Probing

• Labeled probe is added to the membrane

• Probe seeks its complementary target sequence and hybridizes

Step 6: Detection

• Radioisotope label → Autoradiography / X-ray film

• Fluorescent label → Fluorescence microscope

• Non-radioactive label → Chemiluminescence

Radioisotope process mnemonic — BL BWH:
Blotted → Look for sequence → Bind → if not, Wash → Hybridization

NORTHERN BLOT — KEY DIFFERENCES FROM SOUTHERN

Target = RNA (mRNA) | Studies gene expression and RNA structure

STRINGENCY — VERY HIGH YIELD CONCEPT

Stringency = degree of homology (complementarity) required between probe and target.

Why low salt increases stringency:
Salt shields the negative phosphate charges on DNA. Less salt = less shielding = DNA less stable = easier to denature = stricter hybridization conditions.

4 factors that affect stringency: Temperature · Salt concentration · Denaturant concentration · Length and nature of probe

Clinical application: Detecting MRSA or a very specific sequence requires very high stringency — strict conditions, no room for nonspecific binding.

FISH (Fluorescence In Situ Hybridization)

• Same hybridization principle but uses fluorescently labeled probes

• Detects chromosomal abnormalities — translocations, deletions, duplications

• Classic example: Philadelphia chromosome translocation in CML leukemia

• Fluorescing region = abnormal/mutated area of chromosome

• Detected via fluorescence microscope

DNA MICROARRAYS

• Uses treated glass chip instead of nylon/nitrocellulose membrane

• Can screen tens of thousands of targets simultaneously using tiny sample amounts

• Uses automated arrayers

• Key advantage over Dot/Slot Blot: tests multiple gene products at once, not limited to one

TEMPERATURE FORMULA (Tm)

Tm = 81.5°C + 16.6 log M + 0.41(%GC) − 0.61(% formamide) − (600/n)

• RNA probe Tm = higher than DNA

• RNA:DNA hybrid → Tm ↑ by 10–15°C

• RNA:RNA hybrid → Tm ↑ by 20–25°C

• Optimal hybridization temp = ~5°C below Tm

Probe is the limiting reagent during hybridization — sensitivity increases as probe concentration increases. Recommended buffer: 10 mL per 100 cm² of membrane.

QUICK MNEMONICS SUMMARY

LAST-MINUTE MUST-KNOWS

• RNA does NOT need denaturation — it's already single-stranded

• The probe is always labeled; the target never is

• Longer probe = more specific; shorter probe = mutation analysis but noisy

• Electrophoretic transfer is always the fastest blotting method

• Southern uses 10X SSC; Northern uses 20X SSC

• Northern requires RNase-free environment — Southern does not

• Radioisotopes are no longer favored due to hazard and short half-life

• Excess/unbound probe must always be washed off regardless of technique

• Star activity = restriction enzyme cutting at wrong sites under nonstandard conditions

• Smear at bottom of gel = degraded DNA; aggregates at top = incomplete digestion