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 A probe is normally a short sequence of nucleotide bases
that will bind to specific regions of a target sequence of
nucleotides.
 The degree of homology between target and probe results in
stable hybridization.
 Probes can range in size from as short as 10 nuicleotide
bases (molecular weight of 3,300) to as long as 10,000
bases or more (molecular weight of 3,300,000).
 The most common size range for most probes is
between 14 and 40 bases. For statistical uniqueness, a
minimum of 20 nucleotide bases are usually needed
for a probe.
Which probe is best????
Short or long?????

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Sts
StsSts
Sts

Sequence tagged sites (STSs) are short DNA sequences that can be used as genetic markers. STSs were introduced in 1989 as a way to map genes along chromosomes using PCR. They serve as landmarks on physical maps of genomes. STSs are mapped by breaking genomes into fragments, replicating the fragments in bacterial cells to create libraries, and using PCR to determine which fragments contain STSs. Different types of STS markers include microsatellites, SCARs, CAPs, and ISSRs, each of which has distinct characteristics and applications in genetic mapping, population studies, and other areas.

SAGE (Serial analysis of Gene Expression)
SAGE (Serial analysis of Gene Expression)SAGE (Serial analysis of Gene Expression)
SAGE (Serial analysis of Gene Expression)

SAGE (Serial Analysis of Gene Expression) is a technique that allows for the rapid and comprehensive analysis of gene expression patterns in a given cell population. It works by isolating mRNA, synthesizing cDNA, ligating short sequence tags to the cDNA, and then counting the number of times each tag is observed to quantify gene expression levels. The tags are concatenated and sequenced to generate vast amounts of data that must be analyzed computationally to identify which genes particular tags correspond to and to compare expression profiles between cell types. SAGE provides an overview of a cell's complete transcriptional activity and has been applied to study differences in cancer vs normal cells and to identify targets of oncogenes and tumor suppressor genes.

Artificial chromosomes - YAC and BAC
Artificial chromosomes - YAC and BACArtificial chromosomes - YAC and BAC
Artificial chromosomes - YAC and BAC

This document discusses yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs). YACs are engineered chromosomes derived from yeast DNA that can clone very large DNA sequences in yeast cells of up to 1 megabase. BACs are cloning vectors derived from bacterial DNA that can clone DNA fragments of up to 300 kilobases in E. coli. Both systems allow cloning and propagation of large DNA fragments, but YACs can hold more DNA while BACs are more stable and better for functional analysis in mammalian cells.

sciencebiologygenetic engineering
 Short probes tend to hybridize nucleic acids at very
high rates (in minutes).
 where as longer probes may require reaction times of
hours to achieve a stable hybridization.
 Short probes do have some disadvantages.
 They are subject to more nonspecific hybridizations ,
are limited in specificity , and are more difficult to
label.
 Long probes hybridize more stably than short probes
at high temperatures and low salt concentrations(low
stringency).
- A nucleic acid probe
Is a short , single- stranded molecule of
radioactively labeled or fluorescently labeled DNA or
RNA.
 The base sequence of the probe and the conditions
under which the probe is used determine its specificity.
 What ever label is used, it has to be attached to, or
incorporated into, the nucleic acid probe. Here are 5
methods of labelling probes.
1) Nick translation
2) Primer extension
3) RNA polymerase
4) End labelling
5) Direct labelling

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Site directed mutagenesis
Site  directed mutagenesisSite  directed mutagenesis
Site directed mutagenesis

Site-directed mutagenesis is a technique used to introduce specific changes to the DNA sequence of a gene by altering the nucleotide sequence. It allows researchers to study the impact of mutations by changing individual bases, deleting bases, or inserting new bases. There are different methods of site-directed mutagenesis including oligonucleotide-based methods and PCR-based methods. Site-directed mutagenesis has applications in research, production of desired proteins, and development of engineered proteins for commercial uses like detergents.

recombinant dna technologytransgenesmutagenesis
Chromosome walking
Chromosome walkingChromosome walking
Chromosome walking

Chromosome walking is a technique used to analyze large regions of DNA by identifying overlapping clones. It involves selecting a starting clone, subcloning fragments from the ends, and using those fragments to screen a genomic library and identify neighboring clones with overlapping sequences. This process is repeated to "walk" along the chromosome. Chromosome walking was developed in the 1980s and is useful for finding mutations associated with genetic diseases and detecting single nucleotide polymorphisms, though it has limitations with repetitive sequences.

M13 phage
M13 phageM13 phage
M13 phage

Modified M13 vectors have a large number of cloning sites which allow for insertion of foreign DNA. These vectors are derived from the M13 bacteriophage and are commonly used for DNA sequencing, mapping and mutagenesis experiments in molecular biology research. The document appears to be a seminar topic submission about using the M13 phage for biotechnology applications.

 It involves introducing single- strand breaks (nicks by
endonuclease) in the DNA, leaving exposed 3’ hydroxyl
termini and 5’ phosphate termini.
 Nick serves as a start point for introducing new
nucleotides at the 3’ hydroxyl side using DNA
polymerase.
 As a result, the nick will be moved progressively along
the DNA (translated’) in the 5’ 3’ direction.
 The synthesis reaction allows the incorporation of
labelled nucleotides in place of the previously existing
unlabeled.
Labelling of dna
 Based on hybridization of a mixture of all possible
hexanucleotides.
 The starting DNA is denatured and then cooled slowly
so that the individual hexanucleotides can bind to
suitably complementary sequences within the DNA
strands.
 DNA synthesis occurs in the presence of the four
dNTPs, at least one of which has a labelled group.
Labelling of dna

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This document discusses several methods for selecting recombinant clones after introducing recombinant DNA into host cells: - Direct selection involves using a gene from the inserted DNA that confers antibiotic resistance to select clones that grow on media containing that antibiotic. - Insertional inactivation selection works by inactivating a host gene when foreign DNA inserts into it, allowing selection of recombinants. - Blue-white screening uses a vector with a disrupted lacZ gene; foreign DNA insertion repairs the gene, allowing recombinants to be identified by colony color. - Colony hybridization detects recombinants by transferring colonies to a membrane and probing for the inserted DNA sequence. - Immunological tests identify clones expressing antigens encoded by the

sciencerdna technologyplasmids
Scoring matrices
Scoring matricesScoring matrices
Scoring matrices

Scoring system is a set of values for qualifying the set of one residue being substituted by another in an alignment. It is also known as substitution matrix. Scoring matrix of nucleotide is relatively simple. A positive value or a high score is given for a match & negative value or a low score is given for a mismatch. Scoring matrices for amino acids are more complicated because scoring has to reflect the physicochemical properties of amino acid residues.

Whole genome shotgun sequencing
Whole genome shotgun sequencingWhole genome shotgun sequencing
Whole genome shotgun sequencing

Whole genome shotgun sequencing involves randomly breaking genomic DNA into small fragments, sequencing the fragments, and then reassembling the sequences using overlapping regions. The document outlines the history and procedure of shotgun sequencing. Genomic DNA is first fragmented, end-repaired, and size-selected into small, medium, and large fragments. Libraries are created for each size fragment and sequenced. A base caller filters poor calls and an assembler finds overlaps to generate continuous nucleotide sequences or contigs of the whole genome.

shotgunshotgun sequencingsanger sequencing
 DNA of interest is denatured to give single-strands.
 Random primer sequences are added (or sequences
unique to a particular sequences of interest).
 DNA polymerase is added together with labelled
nucleotides.
 Complementary DNA strands are synthesized starting
from the primer sequences and incorporating the
labelled nucleotides. There is partial filling in of the
gaps between the primers.
 DNA is then denatured to release the labelled probe
molecules.
Labelling of dna
 Single-stranded oligonucleotides are usually end-labelled
using polynucleotide kinase ( kinase end- labelling).
 Label is provided in the from of a 32P at the gama-
phosphate position of ATP and the polynucleotide kinase
catalyses an exchange reaction with the 5’-terminal
phosphates.
 The same procedure can also be used for labelling double-
stranded DNA.
 Here fragments carrying label at one end only can then be
generated by cleavage at an internal restriction site,
generating two differently sized fragments which can be
separated by gel electrophoresis and purified.
Labelling of dna

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Colony hybridisation
Colony hybridisation Colony hybridisation
Colony hybridisation

This document summarizes the process of colony hybridization. Colony hybridization allows researchers to select bacterial colonies containing specific genes. The procedure involves lysing bacterial colonies on a nitrocellulose filter, denaturing the DNA, and hybridizing the DNA to a labeled probe for the target gene. Unbound probe is then washed away. Where the probe binds to colonies containing the target gene, dark spots will appear on an x-ray film placed over the filter, allowing identification of recombinant colonies containing the desired gene. Colony hybridization has applications in identifying recombinant bacteria, cytogenetics studies, disease diagnosis, fingerprinting, and screening bacterial colonies.

sciencebiotechnologymicrobiology
Phage display
Phage displayPhage display
Phage display

Phage display technology allows the display of proteins or peptides on the outside of bacteriophages while encoding the corresponding gene on the inside. This allows for large libraries to be screened in vitro to select for interactions between the displayed molecules and a target. The most common phages used are filamentous phages like M13, which can be genetically manipulated to display proteins of interest. The technique involves inserting a gene into the phage coat protein gene, infecting bacteria to produce phage particles displaying the protein, and panning against a target to isolate interacting proteins.

phage disply technology
Linker, Adaptor, Homopolymeric Tailing & Terminal Transferase
Linker, Adaptor, Homopolymeric Tailing & Terminal TransferaseLinker, Adaptor, Homopolymeric Tailing & Terminal Transferase
Linker, Adaptor, Homopolymeric Tailing & Terminal Transferase

A brief idea about the problems and solutions in cloning regarding Linker, Adaptor, Homopolymeric Tailing & Terminal Transferase enzyme.

biotechnologycloninglinkers
o The preparation of labelled RNA probes (riboprobes) is
most easily achieved by in vitro transcription of insert DNA
cloned in a suitable plasmid vector.
o The vector is designed so that adjacent to the multiple
cloning site is a phage promoter sequence , which can be
recognized by the corresponding phage RNA polymerase.
o By using a mix of NTPs , high specific activity radiolabeled
transcripts can be generated.
o Labelled sense and antisense riboprobes can be generated
from any gene cloned in such vectors and are widely used
in tissue in situ hybridization.
o DNA template + RNA polymerase + labelled
ribonucleotides ======= labelled RNA probe.
Labelling of dna
 Radiolabels
 Non-radioactive labels
 Chemiluminescence
 Fluorescence
 Antibodies
 Probe nucleic acid can be labelled using radioactive isotopes,
e.g 32p, 35S ,125I ,3 H.
 Detection is by autoradiography or geiger-mullur counters.
 Radiolabelled probes used to be the most commen type but
are less popular today because of safety considerations.
 However,radiolabelled probes are the most sensitive ,e.g .32p
labelled probes can detect single –copy genes in only 0.5 mg
of DNA.
 High sensitivity means that low concentrations of probe –
target hybrid can be detected.

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Electrophoretic mobility shift assay

This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.

#science#proteomics#research
Yeast artificial chromosomes
Yeast artificial chromosomesYeast artificial chromosomes
Yeast artificial chromosomes

Artificial chromosomes are synthetic chromosomes introduced into host cells to propagate and transfect DNA fragments larger than plasmids can hold. Yeast artificial chromosomes (YACs) specifically are engineered chromosomes derived from yeast DNA ligated into bacterial plasmids, allowing insertion of 100-1000kb DNA fragments. YACs contain elements for yeast and bacterial replication and selection, and are useful for cloning large genomic fragments like whole human genes for mapping the genome.

yeast artificial chromosomes
Complementary DNA (cDNA) Libraries
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Complementary DNA (cDNA) Libraries

This document discusses different strategies for cloning DNA fragments from complex sources like genomic DNA or cDNA. There are two major approaches - cell-based cloning, which divides the DNA into fragments that are cloned to create a library, and directly amplifying target sequences using PCR. The document focuses on cDNA library construction, explaining that cDNA libraries reveal gene expression profiles. It describes early cDNA cloning methods and their limitations, as well as improved directional and non-directional cloning techniques. Finally, it discusses various screening methods for identifying clones of interest from cDNA libraries, including colony hybridization, plaque lifts and immunological screening.

molecular biologycdna libraries
 These are harmless than radiolabels and do not require
dedicated rooms , glassware and equipment or staff
monitoring , etc.but they are not generally as sensitive.
 Some examples:
 Biotin this labels can be detected using avidin or
streptavidin which have high affinities for biotin .
 Enzymes the enzymes is attached to the probe and its
presence usually detected by reaction with a substrate that
changes colour .Used in this way the enzymes is sometimes
referred to as a “reporter group”.
 Examples of enzymes used include alkaline phosphatase
and horseradish peroxidase.
 In this method chemiluminescent chemicals attached
to the probe are detected by their light emission
using a luminometer.
 Fluorescence chemicals attached to probe fluorescence
under UV light. This type of label is especially useful for
the direct examination of microbiological or cytological
specimens under the microscope- a technique known
as fluorescent in situ hybridization (FISH).
Labelling of dna
 Antibodies an antigenic group is coupled to the probe
and its presence detected using specific antibodies .
Also ,monoclonal antibodies have been developed that
will recognize DNA-RNA hybrids.

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Genomic library constructionGenomic library construction
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A DNA library is a collection of DNA fragments that have been cloned into vectors. DNA libraries allow researchers to isolate and study specific DNA fragments of interest. To create a genomic library, DNA is extracted from an organism, cut into fragments, inserted into vectors, and introduced into host bacteria to generate clones containing all the organism's DNA sequences. This library can then be screened to identify and study particular genes. DNA libraries provide an efficient way to store, isolate, and analyze DNA sequences.

dnacdnarna
S1 Mapping
S1 Mapping  S1 Mapping
S1 Mapping

S1 Mapping is a laboratory method used for locating the start and end points of transcripts and for mapping introns. This technique is used for quantifying the amount of mRNA transcripts, it can therefore identify the level of transcription of the gene in the cell at a given time.

biologybiotechnologymolecular biology
Nucleic acid probes
Nucleic acid probesNucleic acid probes
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A probe is a short sequence of DNA or RNA that is used to detect complementary DNA or RNA sequences in samples. Probes can be labeled with radioactive isotopes or fluorescent tags to allow for their detection after hybridizing to target sequences. They are commonly used techniques like Southern blots, Northern blots, and in situ hybridization to detect specific nucleic acid sequences and identify microorganisms, viruses, or genetic mutations associated with diseases. Probes provide a sensitive method for detecting nucleic acids and have many applications in medical research and diagnosis.

nucleic acid probes
 Probe and target hybridize with each other but how
this is brought about can vary. There are 4 main
formats;
 Target (usually) is bound to a solid support such as a
microtitre tray or filter membrane.
 Probe is added in solution and binds to target (if
present ) on solid support.
 After washing to remove unbound probe, hybridization
is detected on the solid support using whatever
method is appropriate for the probe label.
 Both the probe and the target are in solution. Because
both are free to move , the changes of reaction are
maximized and , therefore , this format is generally
faster than others.
 In this format probe solution is added to fixed tissues,
sections or smears which are then usually examined under
the microscope.
 The probe label, e.g a fluorescent marker, produces a visible
change in the specimen if the target sequence is present and
hybridization has occurred.
 However, the sensitivity may be low if the amount of target
nucleic acid present in the specimen is low.This can be used
for the gene mapping of chromosomes, and for the detection
of microorgnisms in specimens.

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Northern, southern and western blotting
Northern, southern and western blottingNorthern, southern and western blotting
Northern, southern and western blotting

The document discusses various techniques used for nucleic acid hybridization, including Southern blotting, Northern blotting, dot blot hybridization, and in situ hybridization. Southern blotting involves separating DNA fragments by size, transferring them to a membrane, and using a labeled probe to detect complementary DNA sequences. It can be used to detect mutations. Northern blotting is similar but detects RNA. Dot blot hybridization spots DNA/RNA samples directly onto a membrane. In situ hybridization detects nucleic acids within intact cells using labeled probes. Microarrays allow simultaneous screening of thousands of genes using hybridization on an array.

molecular biologybiotechnology
Blotting techniques
Blotting techniquesBlotting techniques
Blotting techniques

This document discusses various blotting techniques used to detect specific DNA, RNA, and protein molecules. It describes Southern blotting for detecting DNA, Northern blotting for detecting RNA, and Western blotting for detecting proteins. Southern blotting involves separating DNA fragments by gel electrophoresis, transferring them to a membrane, and using a labeled probe for detection. Northern blotting is similar but used for detecting specific RNA sequences. Western blotting uses SDS-PAGE gel electrophoresis to separate proteins, transfers them to a membrane, and detects them using primary and secondary antibodies. These techniques allow detection of specific biomolecules among many contaminants and have various applications in research and diagnostics.

Blotting techniques
Blotting techniquesBlotting techniques
Blotting techniques

This document describes three types of blotting techniques - Southern blotting, Northern blotting, and Western blotting. Southern blotting is used to detect DNA fragments separated by agarose gel electrophoresis. Northern blotting detects specific RNA sequences separated by gel electrophoresis. Western blotting identifies proteins separated by SDS-PAGE gel using an antibody probe. The document provides detailed procedures and applications for each type of blotting.

biochemistry
 After size fractionation of nucleic acids by electrophoresis,
they are transferred to a filter membrane which is then
probed.
 The presence of target is confirmed by detection of probe on
the filter membrane, e.g radiolabelled probe can be detected
by autoradiography and the bands in the original gel
determined.
1.Southern blots:
Detection of gel –fractionated DNA molecules
transferred to a membrane. This includes restriction
fragment length polymorphism (RFLP) analysis.
2.Northern blots:
As above but used for RNA.
3.Dot blots:
Detection of unfractionated nucleic acid
immobilized on a membrane.
4. Colony and plaque blots:
Detection of immobilized nucleic acid on a
membrane that has been released from lysed bacteria
or phages.
THANK YOU

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Labelling of dna

  • 1. V.SNEGA I M.sc BIOTECHNOLOGY BON SECOURS COLLEGE FOR WOMEN THANJAVUR
  • 2.  A probe is normally a short sequence of nucleotide bases that will bind to specific regions of a target sequence of nucleotides.  The degree of homology between target and probe results in stable hybridization.
  • 3.  Probes can range in size from as short as 10 nuicleotide bases (molecular weight of 3,300) to as long as 10,000 bases or more (molecular weight of 3,300,000).  The most common size range for most probes is between 14 and 40 bases. For statistical uniqueness, a minimum of 20 nucleotide bases are usually needed for a probe.
  • 4. Which probe is best???? Short or long?????
  • 5.  Short probes tend to hybridize nucleic acids at very high rates (in minutes).  where as longer probes may require reaction times of hours to achieve a stable hybridization.  Short probes do have some disadvantages.  They are subject to more nonspecific hybridizations , are limited in specificity , and are more difficult to label.  Long probes hybridize more stably than short probes at high temperatures and low salt concentrations(low stringency).
  • 6. - A nucleic acid probe Is a short , single- stranded molecule of radioactively labeled or fluorescently labeled DNA or RNA.
  • 7.  The base sequence of the probe and the conditions under which the probe is used determine its specificity.
  • 8.  What ever label is used, it has to be attached to, or incorporated into, the nucleic acid probe. Here are 5 methods of labelling probes. 1) Nick translation 2) Primer extension 3) RNA polymerase 4) End labelling 5) Direct labelling
  • 9.  It involves introducing single- strand breaks (nicks by endonuclease) in the DNA, leaving exposed 3’ hydroxyl termini and 5’ phosphate termini.  Nick serves as a start point for introducing new nucleotides at the 3’ hydroxyl side using DNA polymerase.  As a result, the nick will be moved progressively along the DNA (translated’) in the 5’ 3’ direction.  The synthesis reaction allows the incorporation of labelled nucleotides in place of the previously existing unlabeled.
  • 11.  Based on hybridization of a mixture of all possible hexanucleotides.  The starting DNA is denatured and then cooled slowly so that the individual hexanucleotides can bind to suitably complementary sequences within the DNA strands.  DNA synthesis occurs in the presence of the four dNTPs, at least one of which has a labelled group.
  • 13.  DNA of interest is denatured to give single-strands.  Random primer sequences are added (or sequences unique to a particular sequences of interest).  DNA polymerase is added together with labelled nucleotides.  Complementary DNA strands are synthesized starting from the primer sequences and incorporating the labelled nucleotides. There is partial filling in of the gaps between the primers.  DNA is then denatured to release the labelled probe molecules.
  • 15.  Single-stranded oligonucleotides are usually end-labelled using polynucleotide kinase ( kinase end- labelling).  Label is provided in the from of a 32P at the gama- phosphate position of ATP and the polynucleotide kinase catalyses an exchange reaction with the 5’-terminal phosphates.  The same procedure can also be used for labelling double- stranded DNA.  Here fragments carrying label at one end only can then be generated by cleavage at an internal restriction site, generating two differently sized fragments which can be separated by gel electrophoresis and purified.
  • 17. o The preparation of labelled RNA probes (riboprobes) is most easily achieved by in vitro transcription of insert DNA cloned in a suitable plasmid vector. o The vector is designed so that adjacent to the multiple cloning site is a phage promoter sequence , which can be recognized by the corresponding phage RNA polymerase. o By using a mix of NTPs , high specific activity radiolabeled transcripts can be generated. o Labelled sense and antisense riboprobes can be generated from any gene cloned in such vectors and are widely used in tissue in situ hybridization. o DNA template + RNA polymerase + labelled ribonucleotides ======= labelled RNA probe.
  • 19.  Radiolabels  Non-radioactive labels  Chemiluminescence  Fluorescence  Antibodies
  • 20.  Probe nucleic acid can be labelled using radioactive isotopes, e.g 32p, 35S ,125I ,3 H.  Detection is by autoradiography or geiger-mullur counters.  Radiolabelled probes used to be the most commen type but are less popular today because of safety considerations.  However,radiolabelled probes are the most sensitive ,e.g .32p labelled probes can detect single –copy genes in only 0.5 mg of DNA.  High sensitivity means that low concentrations of probe – target hybrid can be detected.
  • 21.  These are harmless than radiolabels and do not require dedicated rooms , glassware and equipment or staff monitoring , etc.but they are not generally as sensitive.  Some examples:  Biotin this labels can be detected using avidin or streptavidin which have high affinities for biotin .  Enzymes the enzymes is attached to the probe and its presence usually detected by reaction with a substrate that changes colour .Used in this way the enzymes is sometimes referred to as a “reporter group”.  Examples of enzymes used include alkaline phosphatase and horseradish peroxidase.
  • 22.  In this method chemiluminescent chemicals attached to the probe are detected by their light emission using a luminometer.  Fluorescence chemicals attached to probe fluorescence under UV light. This type of label is especially useful for the direct examination of microbiological or cytological specimens under the microscope- a technique known as fluorescent in situ hybridization (FISH).
  • 24.  Antibodies an antigenic group is coupled to the probe and its presence detected using specific antibodies . Also ,monoclonal antibodies have been developed that will recognize DNA-RNA hybrids.
  • 25.  Probe and target hybridize with each other but how this is brought about can vary. There are 4 main formats;
  • 26.  Target (usually) is bound to a solid support such as a microtitre tray or filter membrane.  Probe is added in solution and binds to target (if present ) on solid support.  After washing to remove unbound probe, hybridization is detected on the solid support using whatever method is appropriate for the probe label.
  • 27.  Both the probe and the target are in solution. Because both are free to move , the changes of reaction are maximized and , therefore , this format is generally faster than others.
  • 28.  In this format probe solution is added to fixed tissues, sections or smears which are then usually examined under the microscope.  The probe label, e.g a fluorescent marker, produces a visible change in the specimen if the target sequence is present and hybridization has occurred.  However, the sensitivity may be low if the amount of target nucleic acid present in the specimen is low.This can be used for the gene mapping of chromosomes, and for the detection of microorgnisms in specimens.
  • 29.  After size fractionation of nucleic acids by electrophoresis, they are transferred to a filter membrane which is then probed.  The presence of target is confirmed by detection of probe on the filter membrane, e.g radiolabelled probe can be detected by autoradiography and the bands in the original gel determined.
  • 30. 1.Southern blots: Detection of gel –fractionated DNA molecules transferred to a membrane. This includes restriction fragment length polymorphism (RFLP) analysis. 2.Northern blots: As above but used for RNA. 3.Dot blots: Detection of unfractionated nucleic acid immobilized on a membrane. 4. Colony and plaque blots: Detection of immobilized nucleic acid on a membrane that has been released from lysed bacteria or phages.