DNA on Michael’s Domain

The human genome was just completed (again)

Mon, 15 May 2023 00:00:00 +0000

More than 20 years ago, the human genome was

completed

(Lander et al., 2001) by the Genome Reference Consortium (GRC). This assembly is known as the GRCh38 reference sequence. Although it is based on DNA from an anonymous group of donors, ⅔ of its sequence is derived from one single male donor of African-European ancestry (Genome Reference Consortium, 2023).*

Suojaus UV-valolta

Thu, 12 Dec 2019 00:00:00 +0000

** UV valot luokittellan seuravasti UV-C (100 / 200-280 / 290 nm, lyhytaalto, kova UV)UV-B (290-315 nm, keskiaalto, keskimääräinen UV)UV-A (315-400 nm, pitkäaallon UV, pehmeä UV, “musta valo”)Erityisesti UVC:n suhteen käytetään usein erilaisia aallonpituusrajoja eri UV-tyyppien välisten rajojen määrittelemiseen. Alle 200 nm:n UVC:n aallonpituudet kutsutaan myös “vakuumi-UV (VUV)”. Toiset erottavat UVC-spektrin ja viittaavat aallonpituuksiin välillä 10 - 200 nm “UVC-VUV”. “Extreeminen (äärimmäinen) UV (EUV)” tarkoittaa aallonpituuksia välillä 10-121 nm, ja tämän alueen lyhyessä päässä säteilyä pidetään ionisoivana (niin kuin röntgensäteilyä). En kuitenkaan tiedä mitään selkeää aallonpituusrajaa, jota käytetään ionisoivan ja ei-ionisoivan säteilyn erottelun määrittämiseen. Molekyylibiologia Useimpia molekyylibiologian UV-pöytiå käytetään etidiumbromidilla värjätyn DNA:n kuvantamiseen agaroosigeeleissä. Nämä UV-pöydät käyttävät noin 300-nm aallonpituutta (enimmäkseen 302 nm), mutta joillakin on myös pidempi aallonpituusvaihtoehto. Esim. Alpha Innotech/UVP, Inc/Ultra-Violet Products Ltd./Analytik Jena LM-26E UV-pöytää voidaan käyttää aallonpituudella 302 tai 365 nm). Nyrkkisääntö on, että mitä pidempi aallonpituus, sitä vähemmän se vaurioittaa DNA:ta (samalla myös DNA-interkaloidun etidiumbromidin signaali heikkenee). On myös 254-nm UV-lamppuja, mutta ne eivät sovellu DNA-töihin, koska ne aiheuttavat DNA:ssa mutaatiot jo muutamassa sekunnissa. Tämä ei ole yllättävää, koska DNA:n oma absorptiomaksimi on 260 nm ja se tarkoittaa, että DNA absorboi suurimman säteilymäärän. Siis 302 nm on kompromissi herkkyyden ja DNA-vaurioiden välillä.Työskenteleminen UV-pöydän kanssa ei ole vaaratonta, ja olettaisin, että UV-vaara on suurempi kuin etidiumbromiidivärjäyksen vaara. Jotkut tutkijat irrationaalisesta syystä pelkäävät etidiumbromiidia liikaa (https: //bitesizebio.com/95/ethidium-bromide-a-reality-check/, http://rrresearch.fieldofscience.com/2006/10/heresy-about-ethidium-bromide.html , https://blogs.sciencemag.org/pipeline/archives/2016/04/18/the-myth-of-ethidium-bromide) . Olen nähnyt “auringonpolttaman” yhdessä kollegassani liian pitkältä altistumiselta UV-pöytien UV-valolle. Kasvonaamari suojaa kasvojasi, mutta voit silti polttaa käsiäsi tai dekolteeasi. UV-läpäisevät ja UV-läpinäkymättömät materiaalit Materiaalin (näkyvästä) valon läpinäkyvyydestä ei voida tietää, kuinka tehokkaasti materiaali absorboi UV-valoa. Tavallinen akryylilasi (“Plexiglas”) on läpinäkyvä suuremman aallonpituuden UV-säteilylle (kutsutaan myös UV-A, 315-400 nm) eikä siksi sovellu silmien suojaamiseen ( https://www.gsoptics.com/transmission-curves / ). Akryylilasi voidaan tehdä UV-läpinäkymättömäksi lisäämällä UV-säteilyä absorboivia lisäaineita. UV-suodattavalla akryylilasilla (“museolaadullinen akryyli”) on erilaisia ominaisuuksia. Jos UV-aallonpituus on alle ~ 375 nm, mikä tahansa UV-suodattavaa akryylia käytetään. UF-4-akryylilasi suojaa vähiten: 80% 400 nm: n UV-säteestä johdetaan 2 mm: n levyn läpi. UF-3 suojaa paremmin ja UF-5 absorboi melkein kaiken hyvin näkyvän ultraviolettivalon (> 390 nm). Polykarbonaatti (PC) on ystäväsi Kun tarvitset suojaa UV-säteiltä, polykarbonaatti on kuitenkin ystäväsi. 3 mm paksu polykarbonaatti on käytännöllisesti katsoen täysin läpinäkymätön UV: lle useimmista UV-lähteistä, joita käytetään molekyylibiologiassa 400 nm asti. Siksi UV-suojaavat kasvonaamarit ja aurinkolasit valmistetaan pääasiassa polykarbonaatista.2 mm, joka on hiukan paksumpi kuin polykarbonaattisten aurinkolasien tyypillinen paksuus, on enimmäkseen riittävä, mutta vähemmän tehokas absorptio paksumpiin polykarbonaattiaineisiin verrattuna vain UV: n ollessa yli ~ 385 nm (siis hyvät aurinkolasit suojaavat silmiäsi molekyylibiologian UV-lampuilta , mutta kasvosi iho altistuu silti). Itse asiassa 2 mm paksuissa polykarbonaattisissa aurinkolaseissa on vähemmän kuin 2 mm polykarbonaattia, koska polykarbonaatin molemmilla puolilla on naarmuuntumaton, UV-säteilyä vaimentava pinnoite, koska polykarbonaatti on erittäin pehmeää ja naarmuuntuu helposti. Valitettavasti polykarbonaattimuoveja on vaikea tunnistaa, koska niiden lukumäärä on “7” hartsin tunnistuskoodien (RIC) luettelossa, joka on “Muu” -sekoitettu pussi. Tuottajien tietojen tulkinta **Kun tarkistat lomakkeilla läpinäkyvien materiaalien optiset ominaisuudet, huomaat pian, että niitä on vaikea tulkita. Huomaat pian, että tuottajien verkkosivustojen lähestymistapa on vähemmän tieteellinen, mutta enemmän mainontaa. He puhuvat UV-säteilystä prosenteissa, mutta eivät missään nimessä mainitse materiaalin paksuutta, mikä on yksi tärkeimmistä imeytymisen / läpäisyn näkökohdista ( https://en.wikipedia.org/wiki/Beer%E2%80%93Lambert_law ). Epäilen voimakkaasti, että he käyttävät 2 mm valotieä (paksuus), toinen mahdollisuus on 1 cm (mikä on toinen “vakio” pituus). Jos sinulla on sisäpiiritietoa, ota meihin yhteyttä!

Protection from UV light

Fri, 15 Nov 2019 00:00:00 +0000

First, some definitionsUV-C (100/200-280/290nm, short-wave, hard UV)UV-B (290-315nm, medium-wave, intermediate UV)UV-A (315-400nm, long-wave UV, soft UV, “black light”)Especially for UV-C, different wave-lengths cut-offs are occasionally used to define the borders between the different UV types. Some exclude the wavelengths below 200 nm from UV-C and refer to them with the term “vacuum UV (VUV)”. Others subdevide the UV-C spectrum and refer to the wavelengths between 10 and 200 nm as “UV-C-VUV”). “Extreme UV (EUV)” refers to wave lengths between 10-121 nm and at the short end of this range, radiation is considered to be ionizing (similar to X-rays). However, I do not know of any clear wavelength border that is used to define a separation between ionizing and non-ionizing radiation. Molecular biologyMost UV tables for molecular biology are used to detect ethidium bromid-stained DNA in agarose gels. They use a wavelength around 300nm (mostly 302nm), but some have a longer wavelength option (e.g. the Alpha Innotech LM-26E can be operated at 302 or 365 nm). Rule of thumb is that the longer the wave length the less damage is done to the DNA (but the signal from DNA-intercalated ethedium bromide becomes also weaker). There are 254-nm UV lamps, but these are not suitable for DNA since they will mutate your DNA within seconds. This is not surprising since the absorption maximum of DNA itself is at 260 nm and meaning that the maximum amount of radiation is absorbed by the DNA. Hence the 302 is a compromise between sensitivity and DNA-damage. Working with a UV-table is not without danger and I would assume that there is more danger from UV than from the ethidium bromide stain, which some people (scientists!) for one or the other irrational reason are too much afraid of ( https://bitesizebio.com/95/ethidium-bromide-a-reality-check/ , http://rrresearch.fieldofscience.com/2006/10/heresy-about-ethidium-bromide.html , https://blogs.sciencemag.org/pipeline/archives/2016/04/18/the-myth-of-ethidium-bromide ). I have seen “sunburn” in one of my colleagues from too long exposure to UV light from UV-tables. The face mask protects your face, but you can still burn your arms or your décolletage.UV-transmissive and UV-opaque materialsThere is no way of knowning from the (visible) light transparency of a material how efficiently the material absorps UV light. Regular acrylic glass (“Plexiglas”) is transparent to higher wavelength UV radiation (also called UV-A, 315-400 nm) and is therefore not suitable for protecting the eyes ( https://www.gsoptics.com/transmission-curves/ ). Acrylic glass can be rendered UV-opaque by adding UV-absorbing additives. UV-filtering acrylic glass (“museum grade acrylic”) comes in different qualities. If the UV wave length is below ~375nm, any UV-filtering grade acrylic will do. UF-4 acrylic glass protects the least: 80% of 400nm-UV is passed through a 2mm sheet. UF-3 protects better and UF-5 absorps almost all of the very near-visible light UV (>390nm).Polycarbonate (PC) is your friendHowever, when you need protection from UV, polycarbonate is your friend. 3 mm thick polycarbonate is virtually completely opaque to UV from most UV sources used for molecular biology up to 400 nm. Therefore, UV-protecting face masks and sun glasses are made mostly from polycarbonate.2 mm, which is a bit thicker than the typical thickness of polycarbonate sunglasses, is mostly sufficient but the less efficient absorption compared to thicker polycarbonate matters only for UV above ~385nm (hence, good sun-glasses protect your eyes from molecular biology UV lamps, but your face skin still gets exposed). In fact, 2 mm thick polycarbonate sunglasses have less than 2 mm polycarbonate since they have on both sides of the polycarbonate a non-scratch non-UV-absorbing coating, because polycarbonate is very soft and gets scratched very easily. Unfortunately polycarbonate plastics are difficult to recognize as their number is “7” on the resin identification code (RIC) list, which is the mixed bag of “Other”.Interpreting producers’ dataWhen you check the data sheets for the optical properties of transparent materials, you soon realize that they are difficult to interpret. You soon notice that the approach of producers’ web sites is less scientific, but more advertising. They talk about UV-transmission in %, but do nowhere mention the thickness of the material, which is one of the most important aspects of absorption/transmission ( https://en.wikipedia.org/wiki/Beer%E2%80%93Lambert_law ). I strongly suspect that they use a 2 mm lightpath (thickness), the other possibility being 1 cm (which is the other “standard” length). If you have any insider knowledge, please let me know!

SnapGene and partial restriction digests revisited

Thu, 23 Aug 2018 00:00:00 +0000

Snapgene is a software for the wet lab molecular biologist, who does lots of cloning work (construct design and annotation). Since I last wrote about the SnapGene software ( https://www.snapgene.com/ ), many good things have happened:

Uncertainty about CRISPR's future

Sun, 18 Jun 2017 00:00:00 +0000

Some feared, that the patent decisions on the CRISPR technology this spring might lead to a monopolization of the technology . However, according to a last month’s article in Nature Methods, it is not at all clear at this moment, whether CRISPR will hit a home run for the editing of the human genome. If a single editing event is accompanied by hundreds of unwanted and unpredictable genomic changes, it would be difficult to argue in favor of it due to the unpredictability of the side effects. This is just a single study in mice , but caution is warranted. The Nature Methods article is especially interesting, since the technology just had been used in humans for the first time .Even if the intellectual property is held by a single company, it is unclear, how a patent could be enforced. CRISPR differs from many other technologies by having a very low entry barrier in terms of cost and know-how. Almost every life science researcher could do it at home in their garages…

The Staden package on Ubuntu for bioinformatics dinosaurs

Wed, 27 Jul 2016 00:00:00 +0000

Mostly we use the SnapGene software when we check the sequences of our DNA constructs. However, sometimes SnapGene’s alignment view is not flexible enough and then I fall back to using the ancient Staden Package . I just had upgraded from Ubuntu 14.04 to 16.04 and hence did not have Staden installed. I was pleasantly surprised when the installation of Staden took only about 15 seconds because finally - thanks to the Debian Med team - Staden is available from the universe repository (actually already since October 2014).sudo apt install stadenStaden is clearly not as intuitive as it could be, but it is very powerful and lends itself to automated processing of data. If you have the opportunity to learn it, I would encourage you to do so. The Finnish CSC recorded the Staden course from 2004, in which I participated and you can get the recordings from Funet TV . I just had briefly considered switching from Ubuntu to SuSE because of the ongoing wireless connection debacle that Canonical can’t seem to fix, but considering how non-trivial a manual install of Staden is, this is a big plus for Ubuntu. There are obviously dedicated Linux distributions for bioinformatics purposes, but they all tend to lag behind the latest and greatest developments of the major distros.

Real biohacking

Mon, 23 May 2016 00:00:00 +0000

I have read an interesting blog post by Steven Novella about biohacking . I completely agree that most people that push biohacking are not doing anything, that would justify the use of the term. A new term should be used if you are doing something conceptually new. However, the use of the term biohacking and its relationship to DIY biology and diverse body modification movements has not settled yet.The definition of biohacking as the “freedom to explore biology deeply” makes the term rather meaningless. Hacking as a term has always carried the notion (rightly or not) of doing something illegal or at least borderline legal. To follow this analogy, doping in sports could be considered biohacking (but not drinking coffee - notwithstanding the fact that coffee does fulfil the criteria of doping for some people). Other examples that I would call “true” biohacking would be the use of Crispr/Cas to modify your own DNA. Or developing medical drugs to treat diseases in your own garage.The question is: does anybody do such things? The development of such techniques is highly regulated in both academic research and commercial enterprises and real genetic engineering carries real risks. Not surprisingly, law-enforcement officials are not very happy about still another subculture to watch for signs of bioterrorism . However, the tools are getting easier and easier to use and garage biotech is a real thing (at least Nature thought it is worth a News Feature ). Already a while ago, I did listen to a talk by Thomas Landrain, one of the brains behind the La Paillasse lab (Thomas: you promised that the La Paillasse web pages would be translated into English!), which is a biotech lab that was started with zero money in a garage in a Paris suburb. They are doing quite interesting research (see e.g. this article about his take on DIY biology). There are also conferences about DIY biology: https://www.synenergene.eu/news-item/paris-conference-what-can-do-it-yourself-biology-do . But when does DIY biology become biohacking? Modifying E. coli bacteria to produce erythropoietin and then purifying and injecting it into yourself to increase your sports performance would certainly qualify as biohacking. Imho that example would actually be feasible considering the state of the DIY biology technology…

How much PCR product can you get?

Thu, 10 Mar 2016 00:00:00 +0000

How much PCR product does one get from a typical PCR reaction? 50-120 ng/µl seems to be a typical result, but it very much depends on your PCR conditions. If you need to minimize your primer concentration to maximize specificity, your yields can be significantly below that. Vice versa, if specificity is not an issue (e.g. for some PCR clonings), you can get many times more.Let’s consider the typical maximum amount of a single PCR reaction, which is 100 µl. And let’s assume we do not have specificity issues and therefore we can use large amounts of primer (1 µM each) and dNTPs (0.2 µM). Since the synthesis of every molecule of double-stranded PCR product consumes one primer, the theoretical maximal molar concentration of double-stranded (ds) DNA product is the same as your primer concentration: 1 µM. 1 µM dsDNA would equal 100 pmol for a 100 µl PCR reaction. How much is that in micrograms? That is of course dependent on the length of your PCR product: e.g. 100 pmol dsDNA of 1000 bp is equal to 66 µg.Can you really get that much? Not in our example of a 1000-bp-product. The reason is that the building blocks of the DNA, the dNTPs, become exhausted long before the primers do. For the 1000 bp product, only 40% of the primers are used up when the dNTPs run out (assuming a GC to AT ratio of 50:50 in your amplicon). To make one molecule of a 1000 bp dsPCR product, you need about 2000 molecules of dNTPs. For our example, a 400-bp PCR product would therefore be optimal as both primers and dNTPs get exhausted at the same rate.It seems that if you want to get larger amounts of longer PCR products you would need to increase the dNTP concentration. However, in our example of a 1000 bp product, the theoretical maximal amount of PCR product is about 26µg, which is massive and sufficient for most applications. There is an online calculator, that lets you play around with primer and dNTP concentration and product length: http://www.bioline.com/us/media/calculator/01_14.html

SnapGene - Simply the best DNA manipulation software

Fri, 01 Jan 2016 00:00:00 +0000

Our lab has been using different software packages to plan, document and visualize DNA constructs. Among those that we liked a lot for a long time were Textco’s GeneConstructionKit (GCK) and Clone Manager (Professional) . The latter runs unfortunately only under Windows. However, since several of our computers run Ubuntu Linux , we did run GCK versions 2.5 and 3 using WINE (a compatibility layer that allows us to run native Windows programs under Linux). However, with the upgrade to version 4, GCK became unusably slow under WINE and we were looking for a replacement. We contacted the developers of GCK, but they apparently were either not willing or able to help us. I suppose that the codebase of GCK is probably more than 20 years old and for that reason nobody dares to touch it. Just around that time, SnapGene was released and it fulfilled almost all of our requirements:

DNA base ambiguity codes

Wed, 30 Aug 2006 00:00:00 +0000

Code	Meaning	Bases
A	Adenine	A
C	Cytosine	C
G	Guanine	G
T	Thymine	T
R	puRine	A, G
Y	pYrimidine	C, T
S	Strong (3 H-bonds)	G, C
W	Weak (2 H-bonds)	A, T
K	Keto	G, T
M	aMino	A, C
B	not A	C, G, T
D	not C	A, G, T
H	not G	A, C, T
V	not T	A, C, G
N	Any base	A, C, G, T

Molecular Weight and Extinction Coefficient of Oligonucleotides

Wed, 30 Aug 2006 00:00:00 +0000

The formula to calculate the molecular weight of DNA oligonucleotides is:

MW (g/mol) = (nA × 249,08619) + (nG × 265,0811) + (nC × 225,07496) + (nT × 240,07462)

To calculate ε (epsilon, the extinction coefficient) of an oligo, the formula is: