Reduction in cost of DNA analysis

[CharonZ]

The sequence has to be known beforehand (in this case it is from a SNP or single nucleotide polymorphism database). You won't find novel sequences with this approach but you can track known sequence in samples. The point is that simply by knowing a sequence you won't (usually) know its function. So basically if you got a new loci sequenced you cannot be sure (without further experiments) whether it will lead to a genetic disease or not. The majority of sequences to date (at least in humans) have no experimentally validated functions. These chips allow a high-throughput screening of known sequences and by using correct experimental setups allows the association of certain diseases with certain genetic loci (in this case).

As an example (basically a repetition from above), let's assume you have a disease but you do not know which region is responsible for that. Now you put a large number of sequences derived either from the human genome project or even better, from a database in which a wide collection of loci variations are stored (e.g. in for of SNPs) on a chip. Then you add samples from healthy and ill subjects on that chip. If certain sequences are only found in samples from ill persons, these regions are likely to be associated with the disease.

 

[Billy T]

The sequence has to be known beforehand (in this case it is from a SNP or single nucleotide polymorphism database). You won't find novel sequences with this approach but you can track known sequence in samples. The point is that simply by knowing a sequence you won't (usually) know its function. So basically if you got a new loci sequenced you cannot be sure (without further experiments) whether it will lead to a genetic disease or not. The majority of sequences to date (at least in humans) have no experimentally validated functions. These chips allow a high-throughput screening of known sequences and by using correct experimental setups allows the association of certain diseases with certain genetic loci (in this case).

As an example (basically a repetition from above), let's assume you have a disease but you do not know which region is responsible for that. Now you put a large number of sequences derived either from the human genome project or even better, from a database in which a wide collection of loci variations are stored (e.g. in for of SNPs) on a chip. Then you add samples from healthy and ill subjects on that chip. If certain sequences are only found in samples from ill persons, these regions are likely to be associated with the disease.

Glad to learn that my guess that you would not discover a mutated seequence was correct - I must be starting to understand some of it - thanks again.

ON the central fact of your post I explained to wife some years ago what the "sequencing of human genome" really means with following analogy (which I invented and think accurately conveys the true nature of that):

It is as if we discovered in some cave a huge collection of books all written in a language we can barely understand anything of, but we do understand enough to know that there is a fantastically important collection of knowledge in those books.

 

[CharonZ]

Glad to learn that my guess that you would not discover a mutated seequence was correct

To state it more precisely, one would not discover novel mutated sequences. Remember, the current gene pool is the result of evolution, which already includes a lot of earlier mutations (which then would be present in databases).

 

[Billy T]

To state it more precisely, one would not discover novel mutated sequences. Remember, the current gene pool is the result of evolution, which already includes a lot of earlier mutations (which then would be present in databases).

True. If you go back far enough I think it safe to say: "Humans are 100% mutations." ! Or even worse: All Humans are "mutation monsters."

 

[Billy T]

Hope you are still reading CharonZ

Can you explain following to me in words I can understand?

"Genetic analysis of non-coding genomic regions is fundamental to modern genotyping methodology. What is not appreciated by many, is that the process of analyzing non-coding genomic space (intergenic and untranslated regions, such as introns) for genetic markers and respective haplotypes, was invented well before the term SNP was popularized in the mid 90’s. Genetic Technologies holds patents, filed as early as 1989, which cover the fundamental process of intron analysis. The inventions also include an improved mapping method, which is based on the ability to identify haplotypes of individuals through analysis of non-coding region sequence variation patterns, particularly intron sequence variation patterns. ..."

It is a quote from the CEO of BioSearch. I own shares in Genetic Technologies but not BioSearch, which has just become the first to license Genetic Technologies IP. (They are Austrailian based company that until now mainly had small income from the cattle breading industry in Austrailia, but attracted my attentionas they seem to have some good IP on evaluating the "junk DNA," which everyone now knows is not "junk.")

If you loike read more at:
http://news.morningstar.com/news/ViewNews.asp?article=/BW/20071008006300_univ.xml

BTW the other company discussed in osome posts of this thread ILMN, is doing very well for me - constantly hitting new highs every week, if not every day.

PS anyone else (spuriousM?) who can translate the abouve quote into layman's language, please feel free to do so - I will thank you.

 

[CharonZ]

Maybe a few definitions first:
non-coding genomic space usually refers to DNA regions between genes. However eukaryotes have a mosaic structure in their genes. A part codes for a protein (the exons) a part does not (introns). The latter are thus also non-coding regions.

SNPs or single nucleotide polymorphisms refer to a single base change in a particular location (locus) between members of a species (or in case of non-haploid organisms also at the same locus on different chromosomes). The importance of SNPs is that while these base changes usually do not confer a particular phenotype, they might well be associated with one and can then be used as a genetic marker to track the gene in question.
An example, assume that there is a certain allele associated with breast cancer, but one does not know where it is. One could then compare SNPs of healthy individuals to those with breast cancer and determine whether certain SNPs are overrepresented in the cancer patients. This will ideally give two informations. First you will know which SNPs are associated with breast cancer and secondly you might use the SNP as a starting point to track down genes in its vicinity that might be ultimatively responsible for breast cancer.

A level higher is the haplotype that refers to a given combination of alleles on a chromosome and thus combines many markers (as e.g. several SNPs).

So what the article basically states is that they do not focus on finding SNPs or make haplotype association studies based on SNPs but rather analyze sequence variations in introns and use these as genetic markers. A method that they apparently have patented a while ago.

One has to note that these techniques belong to the field of genotyping and not genome sequencing. In fact the Illumina systems discussed in this thread (both solexa, as well as the chips) were designed for these or similar studies and not for genome sequencing.

 

[Billy T]

Thanks CharonZ for post 26. That was a fast responce - you must lurk al lot.

I think I will be able to under stand more after reading post 26 several times more. May need to get back to you on parts that still are over my head.

Thanks again.

 

[S.A.M.]

"Genetic analysis of non-coding genomic regions is fundamental to modern genotyping methodology. What is not appreciated by many, is that the process of analyzing non-coding genomic space (intergenic and untranslated regions, such as introns) for genetic markers and respective haplotypes, was invented well before the term SNP was popularized in the mid 90’s. Genetic Technologies holds patents, filed as early as 1989, which cover the fundamental process of intron analysis. The inventions also include an improved mapping method, which is based on the ability to identify haplotypes of individuals through analysis of non-coding region sequence variation patterns, particularly intron sequence variation patterns. ..."

There are two types of regions in a genome: coding (makes protein) and noncoding (does not make protein).

DNA is made up of nucleotides (adenine, guanine, cytosine, thymidine). A SNP or single nucleotide polymorphism is where one nucleotide is different from what is usually seen in a single species (or between a pair of chromosomes in an individual), there are several of these and some have been associated with the presence or absence of some diseases and may be either causative or biomarkers


A large portion of the genome is noncoding, and until recently was called junk DNA. Now however, researchers are looking into the role of noncoding DNA in regulating the coding portion of the gene, among other things.


A haplotype for this purpose may be a set of SNPs that are transmitted together and hence, their frequent (or specific) occurrence in subjects with a disease may serve as a marker for that disease. Searching for (mapping) sequence variations (like the haplotype and SNPs) in the noncoding region (in your quote) may help to identify individuals at risk for disease or resistant to some forms of treatment.

e.g. some SNPs identified for colon cancer prevalence

http://nci.nih.gov/cancertopics/understandingcancer/geneticvariation/Slide39

 

[Billy T]

Ok. withhelp of wiki and my unabriged dictionary, I am making some progress on your post 26. The first paragraph was commpletely clear, even with out these aids.
I like that you are careful, but will ignore the parenthetical note about "non-haploid organisms" in second paragraph because, as I understand it, they are organisms that reproduce by parthenogenesis, instead of sexually. (Please correct me whenever I error.) So a SNP is an unusual base pair at some particular location on the DNA, where "unusual" means that most menbers of the species have a certain pairing there but, for example, Tom does not have the pairing that Dick and Harry and most other humans have (or "Spot" differs from most other dogs at that "locus".)

Now next sentence of second paragraph ("The importance of SNPs is ... might well be associated with one and can then be used as a genetic marker to track the gene in question.") leads me to think that the term "SNPs" ONLY refers to small sections of a gene (definitely not the non-gene parts of DNA, but including the "intron parts" of the gene).

From wiki first hit on "allele", I got some idea of what an "allele" is, but I am confused. Is "allele" just another way to refer to the "coding part" of the gene (but not the intron part?), i.e. that sequence of DNA pairings that provides the information for other cells componets to make some protein, or is an "allele" the whole gene? (I have long understood, I think, ideas related to "dominance" etc. of "alleles".) Also, but not very important to me, can one call the sequences of coding DNA of "non-haploid organisms" also "alleles" ?(When the "dominace" characteristic of haploid "alleles" does not exist)?

Your next to last paragraph reinforces the idea that term "SNPs" ONLY refers to the coding part of the gene (not the intron) as it states: "they do not focus on finding SNPs or make haplotype association studies based on SNPs but rather analyze sequence variations in introns..." If this is the case and ILMN and several otrhers are hard at work doing "genotyping" (not genome sequencing) of SNPs, I assume, how can Geneotech have unexpired IP for processing the introns? I.e. is there some characteristic of the intron part of a gene which make ILMN et.al's "genotyping" techniques fail on the intron sections? Or does genetech's IP just cover a different method of doing this "genotyping"? (Which presumably at least BioSearch offers some advantage.)

Thanks again. Also even if not a out right error in the above but from what I stated, you suspect I am very confused still, please try to set me straight.

PS. I do a lot of "teaching" about simple physics here at sciforums, as I enjoy doing that. I hope the same in genetics is true of you. I am your very willing student.

 

[S.A.M.]

“Is "allele" just another way to refer to the "coding part" of the gene (but not the intron part?)”

Suppose you have two DNA fragments -same region of gene from two different individuals -(hypothetical):

ATCCGTCC and ATCCCTCC

there is a difference in a single nucleotide (SNP) and there are two alleles: G and C of that particular fragment of DNA region

 

[Billy T]

Hi SAM. you posted while I was making mine.

....A haplotype for this purpose may be a set of SNPs that are transmitted together and hence, their frequent (or specific) occurrence in subjects with a disease may serve as a marker for that disease. ...

This in agreement, I think, with CharonZ, but I think totally unrelated to the similar "non-haploid organisms." Howerer, as I understand things it would be possible (probably very likely) that "non-haploid organisms" have haplotype sequences in their, I think, single streanded DNA.

As I told CharonZ, if this comment also reflects ignorance on my part, and you can teach me better, please do so.

PS thanks for the illustration of a SNP in post 30, but I did already understand that. I am however not completely clear on whether or not the term "SNP" referes to only the single nucleotide polymorphisms (i.e. C & G in your illustration) or to the small sequence that can be "cut out" from the DNA which contains the single nucleotide polymorphisms.

Also you seem to be using "allele" ("there are two alleles: G and C") differently than I understand it. I think it refers at least to the coding part of a gene (if not the whole gene) and not to only the single nucleotide polymorphisms (the G or C in your illustration)

Perhaps CharonZ can set one (or both?) of us straight one the proper use of "allele." I have already asked him to resolve some of my uncertainity about what an "allele" is and how the term is used.

 

[S.A.M.]

Hi SAM. you posted while I was making mine. This in agreement, I think, with CharonZ, but I think totally unrelated to the similar "non-haploid organisms." Howerer, as I understand things it would be possible (probably very likely) that "non-haploid organisms" have haplotype sequences in their, I think, single streanded DNA.

As I told CharonZ, if this comment also reflects ignorance on my part, and you can teach me beter, please do so.

Some confusion here?

haploid = one set of unpaired chromosomes either egg or sperm, for example, have 23 chromosomes

nonhaploid or diploid = gamete = 23 pairs of chromosomes

A haplotype sequence is simply a combination of alleles at multiple linked locations that are transmitted together. So you are searching for a set rather than a single polymorphism

 

[S.A.M.]

PS thanks for the illustration of a SNP in post 30, but I did already understand that. I am however not completely clear on whether or not the term "SNP" referes to only the single nucleotide polymorphisms (i.e. C & G in your illustration) or to the small sequence that can be "cut out" from the DNA which contains the single nucleotide polymorphisms.

Single Nucleotide Polymorphism

refers to the G and C difference in the DNA fragment of same gene from two individuals in my example

Also you seem to be using "allele" ("there are two alleles: G and C") differently than I understand it. I think it refers at least to the coding part of a gene (if not the whole gene) and not to only the single nucleotide polymorphisms (the G or C in your illustration)

allele refers to the occurences of a DNA region, eg I showed you two occurences of that particular fragment. Traditionally it used to refer only to coding regions but lately it is also used for noncoding. A diploid organism for example, has two alleles, one from each copy (mother and father); when two copies are same it is called homozygous, when two copies are different, it is called heterozygous.

Perhaps CharonZ can set one (or both?) of us straight one the proper use of "allele." I have already asked him to resolve some of my uncertainity about what an "allele" is and how the term is used.

I'm sure he will.

 

[Billy T]

To SAM:

Ok, now I think you did not really mean the SNPs (C & G) alone were "alleles" when you said "there are two alleles: G and C." This is all so new to me that I must read carefully and exactly what you and CharonZ say.

I think I did have some confusion about "non-haploid organisms" based on another error. Namely I thought "haploid" refered to "single stranded DNA" (instead having only one set of chromosomes per cell) I.e. I thought many virus, with only single stranded RNA, were "haploids organisms". Contary to what I said in first post, I now think it is precisely the non-haploids (at least the dipoloids, like humans) organisms that I am interested in.

I do not really know what chromosomes are, except that they are discrete bunches of DNA big enough to be seen with an optical microscope (an proper "staining" I suppose).

 

[S.A.M.]

To SAM:

Ok, now I think you did not really mean the SNPs (C & G) alone were "alleles" when you said "there are two alleles: G and C." This is all so new to me that I must read carefully and exactly what you and CharonZ say.

No I meant that these were two occurrences, i.e. alleles G and C of that particular DNA fragment.

I'm trying to keep it very simple for ease of reading because I am sure you will let me know if it is not clear

About "non haploid" organisms:

SNPs or single nucleotide polymorphisms refer to a single base change in a particular location (locus) between members of a species (or in case of non-haploid organisms also at the same locus on different chromosomes).

I believe CharonZ was referring to the fact that a diploid organism may have a single base difference in the same region of DNA between the two copies of chromosomes

 

[Billy T]

To SAM:

Ok, I am reasonably clear on "non-hapliod organisms" - I got that straight and edited my last post while you were again confirming that you do call the SNPs C & G "alleles." But if one can speak of "dominate" alleles, then surely the allele is not just one location of the base paring along the DNA.

Are you sure that is proper use of "allele" I.e. to refer to a SNP or the more usual "single locus" where there is no polymorphism occuring?

 

[S.A.M.]

I think I did have some confusion about "non-haploid organisms" based on another error. Namely I thought "haploid" refered to "single stranded DNA" (instead having only one set of chromosomes per cell) I.e. I thought many virus, with only single stranded RNA, were "haploids organisms". Contary to what I said in first post, I now think it is precisely the non-haploids (at least the dipoloids, like humans) organisms that I am interested in.

The single stranded organisms are the ones with RNA but not DNA. DNA is double stranded.

I do not really know what chromosomes are, except that they are discrete bunches of DNA big enough to be seen with an optical microscope (an proper "staining" I suppose).

I am a visual person so I will utilise a visual aid to explain this

Nucleotide base pairs form the DNA sequence

A long single piece of DNA bunches together to form chromatid

Two identical chromatids join at the centromere to form a chromosome

So a chromosome is a large molecule made up of two identical strands of a single continuous piece of DNA; it also contains other regulatory elements (like histones) which are required to keep the DNA packaged

 

[Billy T]

To SAM:
First an unimportant note: I think that double stranded RNA dose exist but it is rare. Also think that what makes it still "RNA" is the use of one diferent base than DNA uses.

Thanks for the picture. First time I have seen one that seems to show that chromosones are really made of "coils" of DNA helix. W. & C. got the Noble for that helixs without giving much, if any, credit to the then dead lady who really discorvered it. - She made the x-ray crystal defraction photos they used and many people would immediately recognise that photo implied a helical structure. - You probably know all about her, even remember her hame, which I am ashamed to admit I have forgotten. -IMHO she was "robbed" of the honor she deserved - but what can you do - It is a "man's world." (Old civil rights worker/ leader's POV extends to "Woman's lib" and fact I have only daughters re-inforces that too.)

If one looks carefully at your illustration the location of the "telomere" is indicated too, but it does not seem to be "on the end" of some structure as I though. How is it that it is shortend each replication of the cell (with out losing much more)? Is it sort of like the appendix sticking out from the side of the intestine?

I have a theory that the reason why low calorie diet promotes long life is that the cells divide less frequently so the Telomeres get shorter less often - what do you think of that?

 

[S.A.M.]

To SAM:

Ok, I am reasonably clear on "non-hapliod organisms" - I got that straight and edited my last post while you were again confirming that you do call the SNPs C & G "alleles." But if one can speak of "dominate" alleles, then surely the allele is not just one location of the base paring along the DNA.

Are you sure that is proper use of "allele" I.e. to refer to a SNP or the more usual "single locus" where there is no polymorphism occuring?

The C and G alleles are a way of identifying the two different occurrences of that fragment.

Dominant allele infers the presence of more than one occurrence of an allele in a locus in the same individual and how they interact to produce a phenotype.


e.g. a gene expresses a certain trait like eye color

this gene may have several different loci (singular, locus) with alleles or sections of DNA that contribute to the eye color.

You have two copies of the chromosome (from mother and father) so you have two copies of these alleles.

Now whether your eye color is like mother, father, or some relative on either side will depend on the relative dominance of the alleles from both sides.

 

[S.A.M.]

To SAM:
First an unimportant note: I think that double stranded RNA dose exist but it is rare. Also think that what makes it still "RNA" is the use of one diferent base than DNA uses.

I have heard of it in siRNA but I think it refers to hairpin like RNA rather than two complementary strands.

Thanks for the picture. First time I have seen one that seems to show that chromosones are really made of "coils" of DNA helix (W. & C. got the noble fopr with out giving much if any credit to the then dead lady who really discorvered it. - made the x-rauy crystal defeaction photos that many people whould immediately recognise implied aq helical structure - You probably know all about her, even remember her hame, which I am ashamed to admit I have forgotten -IMHO she was "robbed" of the honor she deserved - but what can you do - It is a "man's world." (Old civil rights worker/ leader's POV extends to "Woman's lib" and fact I have only daughters re-inforces that too.)

Rosalind Franklin.

If one looks carefully at your illustration the location of the "telomere" is indicated too, but it does not seem to be "
"one the end" of some structure as I though. How is it that it is shortend each replication of the cell (with out losing much more)? Is it sort of like the appendix sticking out from the side of the intestine?

The telomere is at the ends of the chromosome and consists of highly repetitive DNA sequences. Every time there is replication, the DNA polymerase does not replicate to the very end, so the telomere is shortened.

I have a theory that the reason why low calorie diet promotes long life is that the cells divide less frequently so the Telomeres get shorter less often - what do you think of that?

Why would a low calorie diet lead to less cell division?