Maarg Manthan · Topic 10.5

DNA Sequencing and Nucleic Acid Probes

Science & Technology › Biotechnology – Principles and Techniques · Topic 10.5

DNA sequencing finds the order of bases in a piece of DNA, while a nucleic acid probe is a short, labelled strand of DNA or RNA that finds and binds a matching sequence by base pairing. Sanger sequencing uses dideoxy chain terminators that stop copying at each base, and the fragments are read to give the sequence. Probes carry a radioactive or fluorescent label and are used in Southern blotting, Northern blotting, in situ hybridisation, DNA microarrays and diagnostic tests. Together these methods allow a scientist to read a gene, to detect a mutation or an infection, and to confirm the identity of a person or a pathogen.

5 min read · UPSC Prelims · Science & Technology · Biotechnology – Principles and Techniques

At a Glance

Sequencing Reads the order of bases
Probe Labelled matching strand
Principle Base pairing
Blotting Southern for DNA, Northern for RNA
Use Diagnosis and forensics
Exam link UPSC Prelims, Science & Technology: Biotechnology – Principles and Techniques

Where Does It Fit?

This topic follows genome sequencing. It moves from the idea of reading whole genomes to the bench methods that read one sequence or look for it with a probe. Previous topic: Genome Sequencing and Its Practical Uses. Next topic: Bioprocess and Fermentation Technology.

How Do Sequencing and Probes Work?

Method	Principle and use
Sanger sequencing	DNA is copied in the presence of chain stopping bases. Fragments of every length are separated and the end base of each is read, giving the sequence
Next generation sequencing	Millions of short fragments are read in parallel and then assembled by computer
Probe hybridisation	A labelled single strand binds a matching sequence in a sample. The label shows where binding has taken place
Southern blotting	DNA fragments are separated, moved to a membrane and tested with a probe. Named after Edwin Southern, 1975
Northern blotting	The same idea for RNA, used to see if a gene is active
Western blotting	Detects proteins with antibodies, as in Antibody-Antigen Interaction and Detection Methods
Fluorescence in situ hybridisation	Fluorescent probes show the position of a gene or chromosome inside cells, used to detect chromosome changes

What Are the Uses of Probes and Sequencing?

Genetic disease testing: probes and sequencing find mutations such as the sickle cell change
Infection tests: probes detect the DNA or RNA of viruses and bacteria
DNA fingerprinting: probes for repeat regions give a pattern that is unique to a person. Alec Jeffreys developed it in 1984
Cancer diagnosis: fluorescence in situ hybridisation finds gene amplification such as HER2 in breast cancer
Gene expression: microarrays and Northern blots show which genes are active
Food and environment: probes detect genetically modified material, pathogens in water and species in food
Research: probes help to locate genes on chromosomes and to clone genes from libraries
Prenatal diagnosis: chromosome probes detect extra or missing chromosomes in fetal cells

What Else Should You Remember?

Complementary pairing: adenine pairs with thymine in DNA and with uracil in RNA, and guanine pairs with cytosine. This is the basis of every probe
Labels: radioactive phosphorus was used earlier, and fluorescent dyes or enzymes are used now for safety
Stringency: the temperature and salt conditions decide how exactly a probe must match before it binds
Microarray: a chip with thousands of fixed probes, used to measure the activity of many genes at once
Padlock and molecular beacon probes: special probes that give a signal only when they bind the correct target
Link with PCR: PCR makes many copies and probes or sequencing then identify them, as in Polymerase Chain Reaction (PCR)
Sanger: Frederick Sanger received two Nobel Prizes in Chemistry, in 1958 and 1980
India: DNA fingerprinting was set up at the Centre for Cellular and Molecular Biology in Hyderabad, with Lalji Singh as a leading scientist
DNA law: the use of DNA profiles in criminal cases is shaped by the laws on DNA technology and forensic rules. Check the latest status of the proposed Bill
Accuracy: false matches are possible if the probe is short or conditions are loose, so controls are always run
Restriction fragment length polymorphism: differences in the length of DNA fragments cut by restriction enzymes, used in early DNA fingerprinting and in gene mapping
Short tandem repeats: short repeated sequences that vary in number between people. Modern forensic profiles compare about twenty such markers
Colony hybridisation: a way to find bacterial colonies that carry a wanted gene by pressing them on a membrane and adding a probe
Reverse transcription: RNA can be changed to DNA before testing, which is how RT-PCR detects RNA viruses

Why Does It Matter for the Exam?

Blot names: Southern for DNA, Northern for RNA, Western for protein
Principle: base pairing and hybridisation
Applications: DNA fingerprinting and disease detection
Current affairs link: forensic DNA use and pathogen testing
Common errors: mixing up the three blots or saying that probes are always radioactive

As an analytical point, probe based tests are specific and fast, which makes them useful in low resource settings. Quality control and trained staff decide whether the result can be trusted.

Why Is This Topic Important?

Techniques Blots and probes
Principle Hybridisation
Uses Diagnosis, forensics

What Came Next?

The next topic covers fermentation and bioprocess technology. Next topic: Bioprocess and Fermentation Technology.

Key People and Terms

Probe Labelled matching strand
Hybridisation Binding of matching strands
Southern blot Detects DNA
Northern blot Detects RNA
FISH Fluorescent chromosome probe
Microarray Chip of many probes

Exam Corner

Points to Remember

Southern blot: DNA; Northern: RNA; Western: protein.
DNA fingerprinting: Alec Jeffreys, 1984.
Sanger: two Nobel Prizes.
Probes work by base pairing.
FISH uses fluorescent probes.

Do Not Confuse With

Southern, Northern and Western blots DNA, RNA and protein
Sequencing and probing Reading an unknown sequence versus finding a known one
Probe and primer Detects a sequence versus starts copying of DNA

Memory Hook

Southern for DNA, Northern for RNA, Western for protein; the probe finds its match.

Mains Angle

Questions ask for the principle and use of each technique. These points help.

Principle Base pairing
Uses Medicine and forensics
Issues Accuracy and privacy

A Question You May Face

An original practice question, not a past paper question.

“Explain the principle of nucleic acid probes and DNA sequencing and discuss their applications in disease diagnosis and forensic science.”

How to Answer

Principle Base pairing.
Methods Sequencing, blots, FISH.
Uses Disease, forensics.
Issues Privacy, quality.

GS Relevance

Prelims: sequencing principle, probes, blotting techniques, applications. GS Paper 3: science and technology, biotechnology, health diagnostics.

Frequently Asked Questions

What is a nucleic acid probe?

A nucleic acid probe is a short single strand of DNA or RNA, labelled with a radioactive or fluorescent marker, that binds to a matching sequence in a sample by base pairing. The label shows where binding has taken place, so probes are used to detect genes, mutations and microbes.

What is the difference between Southern, Northern and Western blotting?

Southern blotting detects specific DNA sequences, Northern blotting detects RNA to show gene activity, and Western blotting detects proteins using antibodies. All three separate molecules by size, transfer them to a membrane and then identify the target. Southern blotting was named after Edwin Southern, who developed it in 1975.

How does Sanger sequencing work?

Sanger sequencing copies a DNA template using normal bases and a small amount of chain stopping bases. This produces fragments that end at every position, and when the fragments are separated by size, the last base of each reads out the sequence. Frederick Sanger developed it in 1977.

What is fluorescence in situ hybridisation?

Fluorescence in situ hybridisation is a method that uses fluorescent probes to find where a particular DNA sequence lies on a chromosome inside cells. It is used to detect extra or missing chromosomes and gene changes in cancer, for example amplification of the HER2 gene in breast cancer.

Who developed DNA fingerprinting?

Alec Jeffreys of the University of Leicester developed DNA fingerprinting in 1984. It uses probes or markers for repeat regions in DNA that differ between individuals, giving a pattern that is almost unique. It is used in criminal investigation, paternity cases and disaster victim identification.

PYQ Practice — Statement Analysis

1 Northern blotting is used to detect RNA.

True

Southern is for DNA.

2 A nucleic acid probe binds to its target by base pairing.

True

Hybridisation.

3 DNA fingerprinting was developed by Frederick Sanger.

False

By Alec Jeffreys in 1984.

4 Western blotting detects DNA sequences.