Gene Probe

Hybridization probe is a fragment of DNA or RNA of variable length (usually 100-1000 bases long), which is used to detect in DNA or RNA samples the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in the probe. The probe thereby hybridizes to single-stranded nucleic acid (DNA or RNA) whose base sequence allows probe-target base pairing due to complementarity between the probe and target. The labeled probe is first denatured (by heating or under alkaline conditions) into single DNA strands and then hybridized to the target DNA (Southern blotting) or RNA (northern blotting) immobilized on a membrane or in situ.


To detect hybridization of the probe to its target sequence, the probe is tagged (or labelled) with a molecular marker; commonly used markers are 32P (a radioactive isotope of phosphorus incorporated into the phosphodiester bond in the probe DNA) or Digoxigenin, which is non-radioactive antibody-based marker. DNA sequences or RNA transcripts that have moderate to high sequence similarity to the probe are then detected by visualizing the hybridized probe via autoradiography or other imaging techniques. Detection of sequences with moderate or high similarity depends on how stringent the hybridization conditions were applied — high stringency, such as high hybridization temperature and low salt in hybridization buffers, permits only hybridization between nucleic acid sequences that are highly similar, whereas low stringency, such as lower temperature and high salt, allows hybridization when the sequences that are less similar. Hybridization probes used in DNA microarrays refer to DNA covalently attached to an inert surface, such as coated glass slides or gene chips, and to which a mobile cDNA target is hybridized.

Depending on the method the probe may be synthesised via phosphoramidite technology or generated and labeled by PCR amplification or cloning (older methods). In order to increase the in vivo stability of the probe RNA is not used, instead RNA analogues may be used, in particular morpholino.












Hope You Like This Post, Let me know what you feel about this blog.
Email me : help.me.ishan@gmail.com

From DNA to RNA

This is the first of two lectures that complete the description of the journey of genetic information from DNA to functional proteins. It begins by describing the key features of how DNA is transcribed into RNA. It then goes on to show how initial transcriptions of RNA are cut up and pasted back together to produce a functional message.Introns are stretches of DNA that lack obvious coding function.Introns are excised from the initial transcript, and the remaining exons are stitched together to produce a contiguous message, leaving much of the original transcript on the cutting-room floor. Armed with this background, we conclude the lecture by revisiting the molecular definition of a gene.
Outline
I The molecular mechanisms of transcription (the synthesis of RNA) are conceptually quite similar to those involved in replication (the synthesis of new DNA), largely because RNA and DNA are very similar molecules.RNA is used as an intermediate, or messenger, passing information from DNA to the protein-synthesis process.


Part 1



Part 2



Part 3






* A The key enzyme involved in transcription is RNA polymerase, which acts much like DNA polymerase. As in replication, the DNA double helix must first unwind, after which RNA polymerase uses complementary base pairing to synthesize a polymer that maintains information as a unique sequence of nucleotide bases.
* B There are some important differences between replication and transcription.


1. RNA polymerase requires fewer enzymes to work than DNA polymerase; for example, RNA polymerase typically opens the double helix itself without the help of helicase.
2. Only one of the two DNA strands is copied during transcription,because only one sequence of nucleotides is needed to code for amino acids.


* The transcribed strand is called the template, or coding strand;the opposite strand is called the complementary strand.
* The template strand, however, is not always on the same side of the double helix, and the distribution of meaningful information on a particular side of the helix is random.
* A complementary strand is unlikely to be a template strand for another gene, because the constraints of complementary base pairing make such compactness difficult.


3 In transcription, only selected pieces of DNA are transcribed, not the entire molecule.

a )Specific nucleotide sequences, called promoters, mark the beginning of transcription for each gene, and other sequences,called termination sites, signal the end of each gene. (Start and stop codons are instructions to the protein synthesizers, not RNA polymerase.)

b)Together, a promoter, termination site, and the DNA in between are called a transcription unit.


4 During replication, only one copy of the DNA molecule is made.During transcription, a single gene may be copied hundreds or thousands of times, depending on the needs of the proteinsynthesis process.
5. RNA polymerase does not on its own recognize promoters;instead, particular stretches of DNA to be transcribed are specified by other proteins that recognize them.
6. The presence or absence of these transcription factors serves as a kind of switch to control when stretches of DNA are to be transcribed into RNA.


II. DNA contains quite a bit of apparently useless material whose purpose is not known.
A. In eukaryotes, the initial RNA transcript includes large segments of RNA that are not part of the code needed to produce a protein. These stretches must be eliminated somehow in order to make a fully functional transcript for the protein.
B. The average size of a transcription unit is about 8,000 nucleotides long.Once the noncoding nucleotides are removed, the average size of a transcript is about 1,200 nucleotides. This means that as much as 85% of the transcribed mRNA is cut out and not translated!

C. The pieces of RNA that are cut out and thrown away are called introns,while the remaining pieces that are spliced together are called exons.
D. The excision of introns and splicing of exons to form a final RNA transcript is done by a set of enzymes and catalytic RNAs known as snRNPs (small nuclear ribonucleoproteins), which act together as a spliceosome. snRNPs identify the ends of introns, break the RNA at these locations while maintaining a connection with the adjacent RNA pieces, then bring these pieces together physically and rejoin the broken strand. The end product is a mature mRNA strand that includes onlyexons.









E. In addition to having introns spliced out, the ends of eukaryotic RNA strands are also chemically modified before the strands are exported from the nucleus.

1. At the 5' end of the polarized RNA molecule, a modified form of guanine is added (the guanine cap); at the 3' end, a string of several hundred adenines (the polyA tail) is added.

2. The guanine cap and polyA tail appear to have two functions. First,they stitch up the ends of the RNA molecule, protecting it from degradation by enzymes that normally break down RNA. Second, they appear to increase the efficiency with which the final RNA transcript is translated into protein after it has left the nucleus.
F. Several hypotheses have been advanced to explain the presence ofintrons.
1. One possibility is that the splicing process controls the rate and/or timing of processing mRNA strands.
2. Introns may also be genetic hitchhikers that serve no purpose and have been introduced into DNA in some way, possibly through retroviruses.
3. In some organisms, the same transcription unit can produce different proteins depending on which parts are excised—one gene’s introns may be another gene’s exons.
4. Many different genes share the same exons, which may be interchangeable structural functions (functional domains), similar to interchangeable machine parts. Introns may be a way of separating common functional domains for easy reuse.