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.












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Dna Array

Pat Brown developed a technique where cDNAs can be embedded onto lass slided.Using these DNA arrays,he could do large-scale expression studies

Growth stage-specific mRNA are isoloated,and then reverse transcribed to give inique cDNA populations.These are directly embedded onto specially cated gass slides.

These slides are coated with poly-lysine,which is positively charged.DNA is negatively charged,so that cDNA "sticks" to the slide through an ionic interaction.The cDNA can still interact with DNA probe.






Brown used srrays to obtain gene expression profiles of cancer.Diffuse large B-cell lymphoma(DLBCL) is a common lymphoma-cancer of the lymp nodes.He found sub-types of DLBCL that correlated with survival rates.

Brown made a chip with genes expressed by the lymph nodes and those importtant in cancer biology .a total of 17856 cDNA genes were printed onto what we called "lymphochip".Remember ,each square on teh chip corresponds to a differnt cDNA.

Then ,he made CDNAs from different DLBCL tumors.

He labelled one set of cDNA with a red florescent tag;the other with a green tag.

Brown incubated the arrays with the tagged cDNAs, which bound to the matching genes printed on the array.

Since he knew the positions of the genes on the DNA array,Brown could figure out the levels of gene expression based on the color signal.If the gene was only expressed in DLBCL1 cells,the square was red.similarly ,of the gene was only expressed in DLBCL2 cells,the square was green.If the gene was expressed equally in both cells,the square was yellow

Thus ,brown identified two sub-types of DLBCL-GC B-like and Activated B-like DLBCL.These sub-types have different responses to the therapy,and with this type of diagnosis,more tailored treatment can begin for patients.This type of tailored treatment is called "pharmacogenomics."

Brown can also analyse the differences in gene expression for these two very similar lymphomas.This may give better understanding of how cancers work, and hopeflly develop beter therapies and cures.














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Microarray Method for Genetic Testing

Genetic testing allows the genetic diagnosis of vulnerabilities to inherited diseases, and can also be used to determine a person's ancestry. Normally, every person carries two copies of every gene, one inherited from their mother, one inherited from their father. The human genome is believed to contain around 20,000 - 25,000 genes. In addition to studying chromosomes to the level of individual genes, genetic testing in a broader sense includes biochemical tests for the possible presence of genetic diseases, or mutant forms of genes associated with increased risk of developing genetic disorders. Genetic testing identifies changes in chromosomes, genes, or proteins. Most of the time, testing is used to find changes that are associated with inherited disorders. The results of a genetic test can confirm or rule out a suspected genetic condition or help determine a person's chance developing or passing on a genetic disorder. Several hundred genetic tests are currently in use, and more are being developed.

What is Southern Blot

Southern blot is a method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample. Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size-separated DNA to a filter membrane for probe hybridization. The method is named after its inventor, the British biologist Edwin Southern.The southern blot is used to verify the presence or absence of a specific nucleotide sequence in the DNA from different sources and to identify the size of the restriction fragment that contains the sequence.
In this procedure, the DNA is isolated from each source and then digested with a specific restriction enzyme. The DNA restriction fragments are then loaded onto an agrose gel and the fragments separated by electrophoresis according to size, with the smaller fragments migrating faster than larger fragments. The DNA is then transferred from the fragile gel to a nylon filter.






Next the radioactively labeled nucleic acid probe is added. The probe binds to complementary DNA segments. Note that the DNA segment being probed is not present in organism B

To detect the position of the radioactive probe, the nylon membrane is covered with an X-ray film. After development, the positions of the probe become visible.

Northern blot

Northern blot is a technique used in molecular biology research to study gene expression. It takes its name from its similarity to the Southern blot technique, named for biologist Edwin Southern. The major difference is that RNA, rather than DNA, is analyzed in the northern blot. Both techniques use electrophoresis and detection with a hybridization probe. The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University.

A northern blot is very similar to a Southern blot except that it is RNA rather than DNA which is extracted, run on a gel and transferred to a filter membrane. There are 3 types of RNA: tRNA (transfer RNA - active in assembly of polypeptide chains), rRNA (ribosomal RNA - part of the structure of ribosomes) and mRNA (messenger RNA - the product of DNA transcription and used for translation of a gene into a protein). It is mRNA which is isolated and hybridized in northern blots.








* mRNA is extracted from the cells grown in galactose and cells grown in glucodse and purified.
* The mRNA is loaded onto a gel for electrophoresis. Lane 1 has gal mRNa Lane 2 has the Glucose mRNA.
* An electric current is passed through the gel and the RNA moves away from the negative electrode. The distance moved depends on the size of the RNA fragment. Since genes are different sizes the size of the mRNAs varies also. This results in a smear on a gel. Standards are used to quantitate the size. The RNA can be visualized by staining first with a fluorescent dye and then lighting with UV.
* RNA is single-stranded, so it can be transferred out of the gel and onto a membrane without any further treatment. The transfer can be done electrically or by capillary action with a high salt solution.
* A GAL DNA probe is incubated with the blot.the single stranded GAL DNA probe binds with immobilized GAL mRNA The blot is washed to remove non-specifically bount probe and then a development step allows visualization of the probe that is bound.

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.


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* 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.

Control of Gene Expression

This lecture begins by illustrating how cells differ in the proteins theye xpress. The same cell may express different proteins at different times in its life, and in multi-cellular organisms, different types of cells typically express different suites of proteins. Given that all cells in an organism have the same set of genes, how do these differences in protein expression arise? The lecture outlines the history of the original experiments done by Jacques Monod and François Jacob in the late 1950s and early 1960s using bacteria to discover the basic mechanisms of gene regulation.


Differences in cell function often produce different cell shapes, but the critical difference is the different protein mix each type of cell contains.

Though many thousands of proteins are coded for in a typical eukaryotic cell, most cells contain only a fraction of that number at any one time. This is the case even in single-celled bacteria. This situation makes sense because making proteins is expensive and many proteins do not store well. This leads to the question of how cells control protein production.
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We know that making proteins requires the transcription of DNA into mRNA, then the translation of mRNA into proteins; thus, we could alternatively ask why all genes in a cell are not always transcribed and translated.

The general answer is that cells regulate gene expression by controlling the conditions necessary for transcription and translation.


The complexity of gene expression means that there are many possible points where cells can control it.

A. In addition to transcription and translation, the initial mRNA transcript must be processed before it is translated, and the final polypeptide must often be further modified by enzymes before it is functional.
B. Cells could theoretically control gene expression at any of these points,but the majority of gene regulation happens at the level of transcription—specifically, by controlling mRNA synthesis.C. This is the case primarily for efficiency; it “costs” cells less to prevent the process from starting in the first place.


III. Control of transcription mechanisms involves genetic “lock-and-key”mechanisms, which work differently in prokaryotes and eukaryotes.

A. To a first approximation, the “switch” that turns transcription on and off is a lock-and-key mechanism.
B. The lock is a specific sequence of nucleotide bases on the DNA that is distinct from but often located physically adjacent to the gene it controls. Each gene has one or more of these regulatory regions, which normally occur “upstream” from the gene.
C. The key is usually a protein with the right shape (including not only physical shape but the correct physical and chemical properties of amino acids) to fit the DNA lock. In general, only one protein will bind to a particular regulatory region.
D. In order to start transcription, RNA polymerase must bind to promoter sites on the DNA molecule.The binding of RNA polymerase to a promoter is not specific; thus, regulatory regions and proteins generally act in two ways to control gene expression.

1. In prokaryotic cells, regulatory regions typically lie in between promoter sites and genes. Regulatory proteins bound to regulatory regions physically block RNA polymerase from reaching the gene.This represents negative control and is typical of prokaryotic cells.
2. The other type of control depends on the fact that RNA polymerase cannot always bind efficiently to the promoter by itself. In this case regulatory proteins nteracting with regulatory regions affect the ability of RNA polymerase to bind to a promoter. In the most general case, regulatory proteins (called “transcription factors”) facilitate the binding of RNA polymerase, resulting in positive control, though these proteins can have either a positive or negative effect on binding in specific cases.
3. These types of controls create two contrasts: physical blockage of RNA polymerase versus an effect on its ability to bind to a promoter region on the DNA, and negative control versus positive control.

E. Though the lock-and-key mechanis m for gene regulation can be compared to turning on a car, there are some important differences.
1. The “driver” (RNA polymerase) is not selective as to which “car” it chooses. RNA polymerase simply tries to transcribe every gene.
2. Some genes will be exp ressed only if the key is not in the regulatory lock.
3. Some genes require a combination of many different keys to be expressed, and some keys must be in their locks, while others must be out.

IV. In the late 1950s and early 1960s, Jacques Monod and François Jacob investigated how gene regulation works in prokaryotes.
A. The bacteria Escherichia coli (E. coli for short) requires several enzymes to metabolize lactose. Biochemists discovered in the early 20th
century that E. coli produces these enzymes only when lactose is available, suggesting that lactose induces enzyme production.
B. This observation led Jacques Monod and François Jacob to the question of how lactose could induce gene expression; they found mutant forms
of E. coli that differed in how they expressed lactose-digesting enzymes.
1. Two kinds of mutants were unable to digest lactose; in one case, an enzyme involved in lactose breakdown itself was defective, while
in another case, an enzyme that brings lactose into the cell was defective. These mutations fit into the expected pattern: A
mutation in a gene causes a coding error that produces a dysfunctional protein, which cannot do its job.
2. A third kind of mutant always produced lactose-digesting enzymes, regardless of whether lactose was present or not. This type of mutation produced a gain of constant function rather than a loss of function. Monod and Jacob inferred that this third type of mutant must have a mutation in some protein involved in
controlling gene expression, not in the genes coding for the enzymes themselves.
C. Monod and Jacob’s idea was revolutionary at the time, because it pointed to a protein whose sole function was to regulate the expression
of other genes.
D. In E. coli, the regulatory protein seemed to inhibit enzyme production.After some debate, physicist Leo Szilard prevailed with the idea that the regulatory protein acted as a repressor that normally preventedproduction of the lactose-digesting enzymes. Szilard’s model suggested that lactose acts as an inducer by disabling the repressor protein; that is,expression of the lactose-digesting enzymes is under negative control.