MUTATIONS and DETECTION OF MUTATIONS USING GOLD NANOPARTICLES

RAHUL MITRAM.Sc ,2nd Semester

Roll No. :13Department Of Materials Science

Outline

• Some basic Terminology used in genetics• What is Mutation• Different causes• Different types of mutations• Effects of Mutations• Several detection techniques• Why Gold Nanoparticles?• Surface plasmon resonance of gold nanoparticles• Detection using GNps• Advantages

Some terminologies• Gametocytes: Cells that take part in reproduction.• Somatic cell: Cells other than gametocytes.• Nucleus: Brain of a cell.• Chromosome: A thread like stucture of nucliec acids and protiens

found in nucleus of most living cells.It carries the genetic information in the form of genes.

• Homologus Chromosome: a couples of homologs are a set of one maternal and one paternal chromosomes that pair up with each other during cell division process.

• DNA: Deoxyribonucleic acid or structural unit of chromosome.• RNA: Ribonucleic acid that takes part in protein synthesis.• Genes: A gene is a locus or regions of DNA which is made up of

nucleotides and it is the molecular unit of heredity.• SPR: Surface plasmon resonance• AuNPs or GNps: Gold nanoparticles.

Structural minimization from cell to genes

What is MUTATION• A sudden change in physical or chemical structure in Genes

or Genetic material.Or more specifically,• Sudden changes in the nucleotide sequence of DNA

• May occur in somatic cells (aren’t passed to offspring).• May occur in gametes (eggs & sperm) and be passed to

offspring.

• The result due to mutation is immediate.

• Mutation was first observed by Hugo de vries in Evening prime Rose (Oenotera lamarkiana) plant.

Causes for MutationThose substances which causes mutation are called Mutagen.

Thee are two types of mutagens:1)Chemical: 3 types:i)Base analogs:Analogus to

Nitrogen Base Pair. E.g-5-Bromo urasil(5-BU)ii)Alkylating Agents.iii)Acridindyes:These are induced mutagens.2)Physical or Radiation: 2 types.

i)Ionizing: This type of mutagens damage the genetic materials by ionizing the atoms present there. E.g.:X-rays,α-rays,β-rays.

ii)Non-ionizing: They do not ionize the molecules in present in the genetic materials. E.g.-UV Rays.

Types of MutationGenerally mutation can divide into 2 main part on basis of where it is occuring-1)Chromosomal Mutation.2)Gene Mutation or Point Mutation.

Chrommosomal Mutation• MORPHOLOGICAL MUTATION:

– May Involve:– Changing the structure of a

chromosome.– The loss or gain of part of a

chromosome.There are 5 types:

i)Deletionii)Duplicationiii)Inversioniv)Translocationv)Non-disjunction

Contd.

Point Mutation• Change of a single nucleotide• Includes the deletion, insertion, or

substitution of ONE nucleotide in a gene• Mainly divided into 3 types:

– 1)Point Mutation

– 2)Inversion

– 3)Frameshift Mutation

Frame Shift Mutation

• Inserting or deleting one or more nucleotides.• Changes the “reading frame” like changing a

sentence.• Proteins built incorrectly.

• There may be 2 chances:– 1)Addition of one N base.– 2)Deletion of one N base.

• Original sequence:– WHY DID THE CAT ATE THE FAT RAT…….

• Deletion of one N-base(Frame shift Mutation):– Let us take Y is being eliminated.– WHD IDT HEC ATA TET HEF ATR AT……

• Addition of one N-base(Frame shift Mutation):– Let us take some P is being added.– WHY PDI DTH ECA TAT ETH EFA TRA T……...

Effect of mutation• A mutation is a permanent change to DNA. These changes

occur when DNA repair mechanisms fail to repair damage, when DNA replicates incorrectly, or from the deletion or insertion of DNA segments. Some mutations occur within the lifetime of a single organism. These somatic mutations involve direct damage to an organism's DNA from an environmental source. For example, damage from ultraviolet rays damages DNA in a way that gives rise to cancerous cells.

• Other mutations are heritable and pass from one generation to another. One particular heritable mutation causes a deletion of 32 base pairs affecting the human CCR5 protein. Research suggests that people with this gene possess immunity to bubonic plague or smallpox, a very beneficial mutation to people at various periods in human history. Because of higher survival rates of people with the CCR5 mutation, the mutation persists into the present day. For modern humans, a CCR5 mutation means resistance to HIV.

Several detection techniques

• Detection of DNA mutations is elementary practice for medical genetic tests as well as for scientific laboratory applications. Medical tests and screens relying on DNA mutation detection include routine preconception and prenatal genetic screens, and tests fordisease predisposition. Traditional methods for mutation detection include sequencing, PCR, fluorescent in situ hybridization, restriction fragment length polymorphism ,biosensors and microarrays.A recent development is detection using nanoparticles.

• Recent studies have demonstrated the use of nanoparticles for DNA mutation detection.Generally they are used as biosensors. These methods are based on nanoparticles that are attached to DNA molecules and change their properties in response to mutations. We study the interaction between DNA molecules and unbound nanoparticles.

• Generaly Gold Nano-Particles are used in detection of SNP(Single Strand Polymorphism) or Point Mutation.

Why Gold Nanoparticles(GNp)?• Within the group of noble metal nanoparticles, gold

nanoparticles are mostly used for biosensor application.• CHARACTERISTICS:

– 1)Gold Nanoparticles are chemically and biologically inert.– 2)These have greater biological compatibility than other nano

particles.– 3)Optical properties like surface plasmon resonance are

fluroscence and chemi-luminescence having better exhibited by Gold Nanoparticles(gold at the nanoscale can appear red, blue, green, or brown).

– 4)They have better stability due to the gold-sulphur bonds.– 5)Production of AuNPs are relatively simple.

SURFACE PLASMON RESONANCE OF GOLD NANOPARTICLES

• Surface plasmon resonance (SPR) is the resonant oscillation of conduction electrons at the interface between a negative and positive permittivity material stimulated by incident light. The resonance condition is established when the frequency of incident photons matches the natural frequency of surface electrons oscillating against the restoring force of positive nuclei.

• The exclusive optical properties of AuNPs arise from localized SPR.

• The oscillation frequency of AuNPs is usually in the visible region, giving rise to a strong color associated specifically with SPR-related absorption.

SPR in AuNPs (left) results in absorbance of blue-green wavelengths and reflection ofred wavelengths, as seen in a photo of a citrate-capped solution of 13-nm-diameter AuNPs(right).

The diameter of AuNPs affects the surface plasmon absorption. 13-nm-diameter AuNPs usually produce an absorbance peak at green-blue wavelengths (~520 nm) and reflect red wavelengths (~700 nm), and such solutions, therefore show a rich red color.

Detection using GNps

• On the basis of these principle we can detect the Point mutation by Colorimetric analysis.

• AuNPs have been used as a colorimetric reporter for SNP sensing based on their unique SPR absorption properties where AuNP aggregation or redispersion of an aggregate triggers a visible color change from PURPLE to RED.

• Free ssDNA can adsorb onto unmodified AuNPs due to the electrostatic effects of nitrogen atoms in the bases, protecting AuNPs from aggregating in a high ionic strength environment. In contrast, dsDNA cannot achieve such protection .Single-base mismatches have been detected by taking advantage of this difference in electrostatic properties between ssDNA and dsDNA.

• At high temperature, the purple color of the perfect match solution would not change to red, whereas a mismatched solution becomes red as the assembled gold nanoparticles separate. Using melting analysis, the position of the point mutation could be identified. This assay provides a convenient colorimetric detection that enables point mutation identification without the need for expensive mass spectrometry.

Advantages• The major advantages of this

technique over the conventional techniques are– Low cost, low sample volume, highly

sensitive and specific AuNP-based colorimetric sensor platforms for the detection of DNA and small molecules.

– This is a naked-eye detection of cancer-related point mutations.

– Need less time(approx. 5mins).

References..• Youtube• Internet

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