Probes are used for hybridization purposes. different types of probes can be used on the basis of what we want to hybridize. May be Radioactive or Non-Radioactive.

1. Nucleic Acid Probes

2. What are Probes?
A small single-stranded nucleic acid sequence(15 - 50 nucleotides) to detect
complementary sequences within a mixture of different nucleic acid sequences.

4. Types of probes
– Single-stranded DNA probes
Most probes are single-stranded nucleic acid fragments
complementary to the gene being sought.
– Degenerate probes
A mixture of all the oligonucleotides that can encode a selected
portion of a peptide sequence
– Oligonucleotide probes
Short synthetic ssDNA
– RNA probes
Generated via in vitro transcription with RNA polymerase
– Antibodies
• Used for “expression” libraries
• Fusion proteins (beta galactosidase + cDNA product)

6. Sources of probes
– Heterologous probes
• From another species (provided genes are highly conserved)
– cDNA probes
• To recover genomic sequences when introns and promoter
elements are needed
– Probe based on protein sequence
• If the amino acid composition of a protein is known, then
degenerate oligonucleotide probes can be generated
• 18-21 bases is sufficient for specific probe (6-7 aa)

7. Preparation of nucleic acid probes:
Conventional DNA probes are isolated by two methods;
1. Cell-based DNA cloning
2. PCR
• In cell-based DNA cloning, the starting DNA may range in size from
0.1 kb to hundreds of kb in length and is usually (but not always)
originally double-stranded.
• PCR-derived DNA probes have often been less than 10 kb long and
are usually, but not always, originally double-stranded.
• RNA probes can conveniently be generated from DNA which has
been cloned in a specialized plasmid vector (which contain a phage
promoter sequence immediately adjacent to the multiple cloning
site).

8. Specificity is important
• Probe must be specific for the gene of interest and should
not anneal to other DNA/RNA sequences that are present
• For most purposes oligonucleotides containing about 20
nucleotides are chosen.
• This is because a specific 20-nucleotide sequence occurs
once in every 420
(≈1012
) nucleotides.
• Since all genomes are much smaller, ≈3 × 109
nucleotides
for humans, a specific 20-nucleotide sequence in a
genome usually occurs only one time.

9. Detection of Probes
Radioactive versus non-radioactive alternatives
– Radioactive: Radioisotopes serve as the tag for identifying
where the probe has bound to desired genomic or cDNA
clones
• Autoradiography required (X-ray film exposed to radioactivity)
– Non-radioactive: Usually based on chemical reactions or
color changes
• Chemiluminescence
• Colorimetric techniques
• Fluorescence (Fluorescence in situ hybridization = FISH

11. Degenerate probes
• In the absence of gene sequence, oligonucleotide probes can be
synthesized from the known amino acid sequence of the protein.
• A 20 oligonucleotide corresponds to approximately 7 amino acid
sequence.
• Usually more protein sequence needs to be determined simply
because many amino acids can be encoded by more than one
codon.
• Due to the degenerate nature of genetic code….rather than a single
oligonucleotide probe, a mixture of oligonucleotides (degenerate
oligonucleotide probe) are synthesized.
• To reduce number of probes its necessary to find a region within the
protein sequence with as little codon degeneracy as possible.
• That is why tryptophan and methionine rich regions are generally
preferred (each has only one codon)

12. 7-19. Designing oligonucleotide probes
based on protein sequence.
The amino acids sequence Cys-Ile-Tyr-Met-His-Gln can be encoded by 48 different
18-base DNA sequences which encode one sequence of amino acids.
48 = 2 × 3 x 2 × 1 × 2 × 2

13. Probe melting temperature (Tm) &
hybridization
Melting temperature (Tm): temperature required to separate double helix
of a DNA fragment into two separate strands
DNA Probes and target DNA are both double stranded, need to separate
before hybridization.
A formula for determining the Tm of the probe fragment is:
Tm = 69.3°C + 0.41[%(G+C)] - 650/l
Where l = the length (in nucleotides) of the probe
So, for a probe of 30 nucleotides with 50% (G+C) content, theoretical Tm
is: (69.3 + 20.5 - 21.7) = 68.1 ° C
Temperature at which hybridization should normally be carried out is
12°C…. below theoretical melting temperature of probe (and target) DNA