Nucleic acid probes - characteristics, probe-assays and applications

Nucleic acid probes are used to detect specific sequence of target DNA or RNA molecules from a mixture.  The single stranded soluble nucleic acid probe hybridizes to the complementary target DNA or RNA that is immobilized on a nitrocellulose or nylon membrane. The nucleic acid hybridization is used for a variety of biotechnological applications such as the detection of cloned DNA, analysis of genetic organization and the diagnosis of genetic diseases. Nucleic acid hybridization is rapid, specific and reliable between complementary strands.

         Definition

In DNA technology, a labelled single stranded nucleic acid molecule is used to tag a specific complementary nucleotide sequence in a nucleic acid sample.

         Kinds of nucleic acid probes

1.    Genomic DNA probes – using probes, more copies of fragments of genomes are obtained, purified and labelled.

2.    cDNA probes – obtained from mRNA using RTase enzyme and labelled.

3.    Synthetic oligonucleotide probes – made up of 14-20 base pairs long.

4.    RNA probes –to locate RNA molecules and was synthesized in vitro using phage RNA polymerase.

Characteristics of DNA probes

1.    DNA probes are very specific in their hybridization wit particular complementary DNA sequence.

2.    DNA probes never produce false positive or false negative results.

3.    DNA probes are more stable and resistant to heat and chemicals. They can be used to characterize DNA from dead tissues.

4.     The size of the DNA probes range from 13 – 100bp.

5.    With the help of PCR, DNA sequences can be amplified up to million folds.

              Labels of DNA probes

·       Labelled with radionuclides-P³², I¹²⁵, S³⁵, H³

·       With fluorescent labels – fluorescein, rhodamines, ethidium, rare earth chelates.

·       With luminescent labels – luminal derivatives, acridium esters, luciferase.

·       With enzyme markers – alkaline phosphatase, horse radish peroxidise.

·       With the combination of above labels

Preparation of DNA probes

The DNA sequence of interest is first cloned into a suitable plasmid. Then the plasmid with the cloned gene is transformed into appropriate host cells to amplify the plasmid. The transformant host cells are then selected using biomarker like antibiotic resistance of plasmids. The transformant host cells are separately cultured to get more copies of plasmid – DNA construct. Then the host bacterial cells are harvested and lysed in order to isolate the plasmid DNA with the cloned gene. The plasmid DNA lysate is precipitated differentially and centrifuged in Cesium chloride density gradients using ethidium bromide to purify plasmid DNA-DNA construct. Plasmid is purified and used for preparing DNA probe by incorporating appropriate label. The cloned gene can be retrieved by REase. The enzyme DNAase is used to cause nicks and DNA polymerase I is used to incorporate radio-labelled (P32 – dCTP) deoxyribonucleotides.  The DNA is denatured using sodium chloride to free out unincorporated deoxyribonucleotides from DNA’ by column chromatography using sephadex 600. Two peaks one with labelled DNA and other for P32 –dCTP are obtained. The first peak is separately collected.

DNA probe assays

The assay consists of 4 steps such as sample preparation, hybridization, separation and detection.

Sample preparation - the samples like blood, CSF, urine, stool or tissues are collected from pathogenic organisms. The separation of the target DNA molecules requires elaborate, time consuming multistep mechanical and chemical extraction procedures. The target molecules may be increased by PCR or LCR etc.

Hybridization – Specific DNA probes are added to the DNA derived from the sample and allowing complementary DNA to join. It may be carried out either as heterogeneous or homogeneous process.

Heterogeneous process uses solid support to immobilize target DNA. Examples are dot blot or in situ hybridization. Homogeneous process occurs in solution and after hybridization event, hybrid DNA is usually immobilized onto a support like filter or beads. Homogeneous process allows rapid and more efficient hybridization.

Separation – column chromatography, electrophoresis or paramagnetic beads may be used to recover true hybrids.

Detection techniques – radioisotopic, flurorescent or chemical luminescent techniques may be used. The probes are appropriately labelled with radioisotopes (I125, P32, S35 or H3) or flurorescent dyes or enzymes.

Applications  of DNA probes

Genetic engineering research - DNA probes are used to identify the clones possessing specific DNA sequences and to identify transformants possessing specific genetic characters. The probes can be used in basic studies in molecular biology, population genetics, and ecological genetics.

Disease diagnosis/epidemiological studies – DNA probes are used to identify genetic or microbial diseases or changes in the DNA of brain tumours.

Identification of food contaminants – it is used to identify pathogenic viruses in imported food samples.

Forensic investigations – Probes are used to identify criminals, solving disputed paternity cases, establishing family relations and solving immigration cases.

Agricultural applications – Probes are used to identify good varieties of seeds or plants.

Taxonomy - Probes are used to identify organisms at species or strain level.

DNA probes are used in anthropological survey of human races, and ante-natal diagnosis of inherited diseases.