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72

One major problem with using uracil as a base is that cytosine can be deaminated, which converts it into uracil. This is not a rare reaction; it happens around 100 times per cell, per day. This is no major problem when using thymine, as the cell can easily recognize that the uracil doesn't belong there and can repair it by substituting it by a cytosine ...


44

The existence of thymine in DNA instead of uracil is apparently due to evolution process which made DNA more stable. Thymine has greater resistance to photochemical mutation, making the genetic message more stable. A rough explanation of why thymine is more protected then uracil, can be found in the article Arthur M, L., Why does DNA contain thymine and ...


22

There is still a lot to be learned about the roles introns play in biological processes, but there are a couple of things that have been pretty well established. Introns enable alternative splicing, which enables a single gene to encode multiple proteins that perform different functions under different conditions. For example, a signal the cell receives ...


14

Thymine has a greater resistance to photochemical mutation, making the genetic message more stable. This offers a rough explanation of why thymine is more protected then uracil. However, the real question is: Why does thymine replace uracil in DNA? The important thing to notice is that while uracil exists as both uridine (U) and deoxy-uridine (dU), ...


14

You need to be careful about everything that comes in contact with your samples. Every spatula, beaker and every magnetic stir bar need to be RNAse-free. For metal- and glassware we usually put everything into a drying closet at 200-250 °C for a few hours. That should get rid of RNAses pretty efficiently. The RNA chemicals should be seperated from other ...


14

Th reason for this is that for the third base of the tRNA non-Watson-Crick pairing is allowed. This phenomenon is called "Wobble base pairing". See the figure (from here) for illustration (from here): If you have a look at the codon table for amino acids, than the variation in the code for one amino acid mostly happens on the third position (from here): ...


13

Here are some tips from what I routinely do: wipe all the surfaces (including pipettes and gloves) with RNAse Away or similar do everything on ice use a RNAse/DNAse free tubes. Use also commercial RNAse free water (it's worth the investment). keep all your RNA reagents separate from your cell culture/animal work reagents. Autoclave all your buffers and ...


13

To make sure I'm not comparing apples and pears, my (attempt to) answer the question will be broken into two parts: comparison of single-stranded nucleic acids and double stranded ones. Single stranded DNA and RNA Both DNA and RNA can form single-stranded complex tertiary structures in which the secondary structure elements are associated through van der ...


11

One of the primary reasons to use poly(A) selection is to eliminate the massive amount of ribosomal RNA (rRNA) present in the samples. The alternative is to use ribosomal RNA depletion kits / techniques to remove as much rRNA as possible before sequencing. Without rRNA depletion a large proportion (~60-80%) of the reads would map to rRNAs. poly(A) ...


10

I don't believe anything should change in the majority of DNA->RNA transcription. DNA methylation typically occurs on the non-watson crick side of Cytosine so it shouldn't affect the base-pairing. However, there are a few hypothetical situations that would result in alterations of the transcribed RNA. The sponatneous deamination of the 4' amine would ...


10

The RNA world hypothesis states that self-replicating RNA (that is, an autocatalytic RNA polymerase) was the first form or precursor of life. So, in that context, your question is basically asking how life originated. The obvious answer is that we don't know (currently anyways), but I'm going to take this opportunity to describe a few really neat experiments ...


10

I think given that you're just getting started with genetics, you can say that the codons are interchangeable. This is generally true, though not technically correct. Here are a few reasons for why this is the case, though there's probably more: Specific organisms use specific codons with different frequencies. This is usually related to the tRNA abundance ...


9

DNA and RNA absorb at 260nm. Proteins absorb at 280nm. The 260/280 ratio is a good estimate of how pure your sample is. For RNA, the 260/280 should be around 2. If it is lower, this might be an indication from contamination or proteins, phenol, or other contaminants in your sample. The 260/230 ratio is a second measure for purity of the sample, as the ...


9

You can clean up phenol by washing with choloroform, and then doing an isopropanol precipitation followed by a 75% EtOH wash (let me know if you'd like an exact protocol). To avoid contamination (and sample loss), you have to be meticulous in your pipetting (which you'll get better with practice). You can always use those phase-lock tubes which basically ...


9

From my understanding sense and anti sense is contextual. If you are looking at a gene from 5'->3'(which is convention) that strand is the sense strand and the complement to the gene is the anti sense strand. However further along the DNA there could be a gene on the 'original' anti sense strand, if you are discussing this new gene, there is a new context ...


9

There are no known natural DNA enzymes (deoxyribozymes), but there are various synthetic DNA enzymes. The first deoxyribozyme that has been reported (Breaker and Joyce, 1994) catalyzed the Pb2+-dependant cleavage of RNA. Various deoxyribozymes have been synthesized, they can catalyze RNA cleavage, RNA ligation and many other reactions like DNA ...


8

The beta-form of DNA and the alpha-form of DNA are based on the pucker on the sugar ribose. As DNA doesn't have a 2'-OH, it can obtain both conformations. RNA does not have this luxury. The steric clash of the 2-OH with the 3'-OH makes the B-form to be very unfavorable thus constraining the RNA to adopt the A-form. Incidentally this steric limitation is ...


8

You should check out Howald C, et al[1]. This is one of the many recent papers tied to the ENCODE data. They've used RT-PCR to amplify exon-exon junctions and then sequenced the results. Supplemental table 2 shows 3076 validated exon-exon junctions in putative processed transcripts which, in the main body of the paper may be sub-classified as: ...


8

The helix shape of DNA molecule is a consequence of its secondary structure. This refers to the bases contained in the molecule which pair, thus determining tertiary structure [1]. Basepairing also occurs in RNA, so it can form a double helix. In fact, RNA is composed of short helices packed together [2]. Base pairs maintain DNA's helical structure no ...


8

See this paper. They have studied RBP-protected sites in the entire human transcriptome by RNA-protein crosslinking followed by RNAse digestion and sequencing: PIPseq. Figure 1 of the paper shows distribution of protein protected sites in RNAs. They also correlate it with different regions of mRNA and its expression. They show number of protein protected ...


8

Nice question which leads to the fundamentals of DNA and RNA. DNA (Deoxyribonucleic acid) is the core of life in Earth, every known living organism is using DNA as their genetic backbone. DNA is so precious and vital to eukaryotes that its kept packaged in cell nucleus, its being copied but never removed because it never leaves the safety of nucleus. DNA ...


8

A pre-tRNA is transcribed from tRNA genes in DNA by RNA polymerase III. Processing occurs in the nucleus, where a 5' sequence is cleaved by RNase P, the 3's CCA motif is added, and ~10% of the nucleotides are substituted. The tRNA are transported out via the pore complexes. Aminoacyl-tRNA synthetase enzymes attach amino acids in the cytoplasm in a 2-step ...


7

I've found that extracted RNA using commercial kits has stayed stable for many years at -80 C. I would certainly aliquot it before freezing however as RNA is particularly sensitive to freeze-thaw cleavage.


7

DNA polymerases need a primer oligonucleotide (RNA or DNA) - their substrates are an existing 3'-OH group and a dNTP. The primase however is a typical RNA polymerase, capable of initiating polynucleotide synthesis de novo by positioning a complementary ribonucleoside 5'-triphosphate opposite its complementary DNA base. The primase makes an RNA primer that ...


7

No, this will not happen. mRNAs are inspected in the nucleus before they are exported into the cytoplasm (at least in eukaryotes), where transcription and translation don't happen at the same place. This ensures that no mRNAs without stop codons or premature stop codons are exported. This phenomenon is called "mRNA surveillance". mRNAs that do not pass this ...


6

This is not my field so I'm risking a wrong/incomplete answer here, but I'd say that the critical difference is the almost complete occurrence of double-stranded DNA that precludes the formation of the tertiary structures in single-stranded RNA, rather than the 2'OH difference. In fact, and following the link you posted, the authors even comment in the ...


6

An ethanol precipitation should work. But I have had great success using the Qiagen RNA cleanup columns, which are in my opinion easier. Here is a URL to see the RNA cleanup columns Qiagen offers: http://www.qiagen.com/products/rnacleanupconcentration/default.aspx Also in the future you should consider using PhaseLock tubes: ...


6

I see big fuzzy bands around 100bp as well. They're most likely RNA contamination. To get rid of them, digest your RT-PCR products with RNAse-H. But if you just need to visualize your band of interest, and the fuzzy bands aren't getting in the way, it shouldn't be a problem. I usually input anywhere from 1-2 ug of RNA into my RT-PCR reaction using the ...


6

Evolution - Douglas J. Futuyma, Chapter 19, p. 461 Michael Lynch and John Conery (2003) have pointed out that a variety of genomic features that appear to have little fitness advantage for organisms-introns, transposable elements, large tracts of noncoding DNA-may be more prevalent in species with small effective population sizes. They have ...


6

DNA-RNA triplex formation is well-documented. It was originally analysed in simple model polynucleotides where the DNA has a polypurine strand and the RNA has a polypyrimidine, e.g. rCCCCCC dGGGGGGGGGGGGGG dCCCCCCCCCCCCCC rUUUUUU dAAAAAAAAAAAAA dTTTTTTTTTTTTT but it is now known to occur in more complex sequences. One of the best studied examples is ...



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