Probe Hybridization and Stringency

Using a labeled probe to find its complement: probe types, the hybridize-then-wash cycle, setting stringency with temperature, salt, and formamide, and the formats - Southern and Northern blots, dot and line blots, in situ hybridization, and FISH with dual-fusion and break-apart probes.

4 min
6 steps
4 questions
Lesson 22 of 60

In this lesson

The idea
Hybridize, then wash
Stringency
Formats
FISH
What to take from this

The idea

A probe is a labeled stretch of nucleic acid that finds its complement by base pairing. Because pairing is specific, a probe picks one sequence out of a whole genome ¹. The base-pairing, melting, and Tm rules are in the Foundations lesson on base pairing.

Probe types:

Probe	Notes
Oligonucleotide (15-50 nt)	synthesized to order; can tell single-base differences apart
Cloned or PCR-made DNA (hundreds of bp to kb)	strong signal; for blots and FISH
RNA (riboprobe)	made by in vitro transcription; RNA:RNA and RNA:DNA hybrids are very stable

Hybridize, then wash

Denature target (and double-stranded probe) to single strands - heat or alkali.
Block the membrane or slide so probe doesn’t stick nonspecifically (salmon sperm DNA, proteins); for genomic probes, unlabeled Cot-1 DNA blocks repetitive sequences.
Hybridize under moderate stringency long enough for probe to find target.
Wash at higher stringency to strip imperfect hybrids.
Detect the label (see Nucleic Acid Labeling).

Stringency

Stringency is how perfect the match must be to survive ¹:

Raise stringency	Lower stringency
higher temperature	lower temperature
lower salt	higher salt
more formamide	less formamide

High stringency keeps only near-perfect hybrids (specific, may lose signal); low stringency tolerates mismatches (sensitive, more background). Labs usually hybridize at moderate stringency and then tune the washes: high background means raise the wash stringency, no signal means lower it.

Quick check

A probe is binding to related, mismatched sequences and giving background. What change raises stringency?

Formats

Format	Target	What it shows
Southern blot	DNA, digested, run on a gel, transferred to a membrane ²	specific fragments and their sizes; still used for large FMR1 expansions and methylation
Northern blot	RNA, run and transferred	transcript presence and size
Dot or slot blot	DNA or RNA spotted directly, no gel	yes/no, semi-quantitative
Line probe (reverse) blot	many probes on a strip, labeled sample hybridized to it	multiplex genotyping, e.g., HPV types or drug-resistance mutations
In situ hybridization (ISH)	nucleic acid in place in fixed cells or tissue	where a sequence is; EBER for EBV, HPV in tumors, kappa/lambda mRNA for clonality
FISH	fluorescent probes on chromosomes or nuclei ¹	translocations, deletions, amplifications, copy number

Name check: the Southern blot is named for Edwin Southern; Northern (RNA) and Western (protein) are lab puns on it.

Quick check

Which blot detects a specific RNA transcript?

FISH

FISH works on metaphase spreads or, more often, interphase nuclei - so it doesn’t need dividing cells and works on tissue sections and smears. A technologist counts colored signals in typically 200 or more nuclei per probe.

Interphase nuclei with fluorescent dots. With dual-fusion BCR and ABL1 probes, a normal nucleus has two red and two green signals and a translocation-positive nucleus has one red, one green, and two yellow fusions. With a break-apart probe, a normal nucleus has two yellow fused signals and a rearranged nucleus has one fused, one red, and one green. — FISH signal patterns. Fusion probes come together when two genes fuse; break-apart probes come apart when one gene is broken. Credit: StudyCorner diagram after Buckingham, Molecular Diagnostics · CC BY 4.0 · Source

Probe design	Normal	Abnormal	Example
Dual-color dual-fusion	2 red, 2 green	1R 1G 2 fusions	BCR::ABL1 t(9;22) in CML
Break-apart	2 fused	1 fused, 1 red, 1 green	ALK in lung cancer, MYC in lymphoma; any partner
Enumeration (centromere)	2 signals	3 (trisomy) or 1 (monosomy)	chromosome 12, 8, X/Y
Locus-specific with control	target = control	target below control (deletion) or far above (amplification)	17p (TP53) deletion in CLL; ERBB2 (HER2) amplification by ratio to CEP17

Quick check

Interphase FISH with BCR::ABL1 dual-fusion probes shows 1 red, 1 green, and 2 yellow signals. This means:

Quick check

Why use a break-apart probe for ALK in lung cancer?

What to take from this

A labeled probe finds its complement; hybridize at moderate stringency, then wash hotter or in lower salt to strip mismatches. Southern blots detect DNA, Northern blots RNA, line blots multiplex genotypes, ISH places sequences in tissue, and FISH counts signals in nuclei. Dual-fusion probes show 1R1G2F for BCR::ABL1; break-apart probes split for any rearrangement of genes like ALK; enumeration and locus probes count chromosomes, deletions, and amplifications.

Lesson complete

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Probe Chemistries and Detection Formats

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Sources for this lesson

1
Lela Buckingham. Molecular Diagnostics: Fundamentals, Methods, and Clinical Applications. 3rd ed. F.A. Davis Company. 2019. verifiedThe standard clinical molecular-diagnostics textbook for MLS/MB programs; author holds MB DLM(ASCP). Covers nucleic-acid chemistry, techniques, lab operations, and applications across infectious disease, oncology, genetics, and identity. Primary topic reference for the ASCP MB program.
2
Michael R. Green, Joseph Sambrook. Molecular Cloning: A Laboratory Manual. 4th ed. Cold Spring Harbor Laboratory Press. 2012. verifiedThe classic three-volume molecular-biology methods manual — authoritative for nucleic-acid isolation, electrophoresis, restriction digestion, labeling, and hybridization techniques. Standard-tier topic reference for the techniques courses.