a genome has been reported. Characterization of an organism by defining every single variant of tens to thousands of rDNA repeat units present in a eukaryotic genome would be quite unreasonable. Here we provide an alternative approach for the characterization of a set of internal transcribed spacer sequences found within every rDNA repeat unit by implementing direct sequencing methodology. The prominent allelic variants and their relative amounts characterizing an individual can be described by a single sequencing electropherogram of the mixed amplicon containing the variants present within the genome. We propose a method for rational analysis of heterogeneity of multicopy genes by compiling a profile based on quantification of different sequence variants of the internal transcribed spacers of the freshwater sponge Ephydatia fluviatilis
Referaat TARTU 2014 Sissejuhatus Esimene täielikult sekveneeritud genoom kuulub bakteriofaagile -X174. Edukas viirusegenoomi DNA järjestuse kindlaks tegemine kannustas ette võtma mahukamaid projekte ja üks kulukamaid nende seas on ,,Inimese genoomi projekt" (HGP, inglise keeles the Human Genome Project). Tänaseks on välja töötatud järgmise põlvkonna sekveneerimismeetodid (NGS, inglise keeles next-generation high-throughput sequencing), mis nõuavad üha vähem ajalisi ja rahalisi ressursse. Selline soodus olukord on vallandanud sekveneerimisbuumi, mis on tõstatanud uue probleemi mida teha saadud andmetega? Üha rohkem tuntakse vajadust sekveneeritud DNA järjestusi analüüsida ja tõlgendada. Paralleelselt sekveneerimismeetodite arenguga proovitakse välja töötada üha täiustatumaid programme andmete töötlemiseks. Spetsiifilisele andmemassiivile sobiva programmi leidmine on oluline ülesanne
*Kordusjärjestuste amplifikatsioon ja lisandumine autosoomidest on arvatavasti päästnud inimese Y kromosoomi. *Mõnedel kukkurloomadel on Y kromosoome limineeritud teatavates somaatilistes kudedes. *Molevoles (Ellobius) on kaotanud Y kromosoomi täielikult. *10 mln.aasta pärast on mees kui selline muutunud ajalooks. *Merohaplodiploidne (XX,X0) soode terminatsioon kujutab endast evolutsiooni lõppmängu Mis eristab meid genoomsel tasandil hiirtest? ·Töö hiire genoomiga Mouse Genome Sequencing Consortium (MGSC). Roti genoomiga RGSC ·Genoomide võrdlus üldiselt: Inimese genoom on veidi suurem, vahe korduselementide hulgast. Hiire intergeensed alad ja intronid (keskmiselt 16%) väiksemad. Esineb lokaalne erinevus. Eksonite ja kodeeriva DNA suurus (550 koodonit) sarnane, sarnane on ka ortoloogsete geenide hulk. G+C hulk hiirel suurem kui inimesel (42% versus 41%). Inimesel palju suurem CpG saarte hulk. Konserveerunud sünteensete piirkondade pikkus 10 Mb
Do not refer blindly to statistics Avoid cliched introductions (Since the old days...) Use quotations or paraphrasing of quotations Linking words Giving examples: For example, for instance, namely Adding information: And, In addition, As well as, Also, Too, Furthermore, Moreover, Apart from, In addition to, Besides Summarising: In short, in brief, in summary, to summarise, in a nutshell (kokkuvõtvalt), to conclude, in conclusion Sequencing ideas: The former, ... the latter, Firstly, secondly, finally, The first point is, Lastly, The following Using linking words is a very good practice to get a good mark! Oral 1. Alguses on sissejuhatav jutt. Ole ilmekas, kasuta head sõnavara. 2. Tõmbad endale teema ja saad ettevalmistusaega 3 minutit. Valmistud monoloogiks ja räägid kokku 2 minutit, ilma vahele segamiseta. o Monoloogi skeem: a) Topic: (minu teema on...)
The main themes that emerged were: • 3D and BIM increase collaboration between different project participants; • A reduction in construction time is evident only when the building models are openly shared; • Intelligent models help to find clashes and reduce re-work; • Models increase accuracy during fabrication and construction; • Shop-drawing review is sped up; • Steel design takes place in a more concurrent fashion; • 3D illustrations help to explain erection sequencing; • Building models provide rigging information for erection crews. The results of this thesis illustrate the benefit that 3D and BIM offer for complex steel construction projects and demonstrate an overall trend in the construction industry. The primary purpose of 3D and BIM is to be able to build the structure in virtual space before actual construction starts, so that the majority of the potential challenges can be successfully identified and addressed during the preconstruction phase.
I am writing with reference to the article you published (in last month's issue). I have just read your article on ... and I feel I must ... You raised some issues which I feel strongly about. At the start of your article, you appear to claim that ... I am afraid I totally disagree. I am completely in agreement. I am sure readers will agree with me when I say that ... ARTICLES Sequencing At first / To start with/In the beginning, ... Then/Next/After that, ... The next thing that happened was ... The next thing I knew was ... Seconds/Minutes later, Late on/Some time later, It wasn't until much later that ... After some time/After what seemed like years, ... Finally/In the end, ... At last, ... Simultaneous events Meanwhile/In the meantime, While all this was going on, In the middle of all this, ... During all this time, ...
dNTP kontsentratsioon: (2/100)*10=0,2 mM/µl Praimerite lõplik kontsentratsioon: (100/100)*5=5 ng/µl 4.5) PCR on eksponentsiaalne. Mitu DNA fragmenti on teoreetiliselt võimalik saada ühest DNA molekulist 5, 15, 30 tsükliga? 5 tsükliga-32 15- 215 30- 230 Praktikas tunduvalt vähem. 14 Töö nr 5: Rekombinantse DNA sekveneerimine ,,DYEnamic ET Terminator Cycle Sequencing Kit" abil. Lähteained: Rekombinantne, inserti sisaldav DNA (~100 ng/l) T7 RNA polümeraasi promootori praimer (100 ng/l) "Sequencing reagent premix" (Amersham), sisaldab fluorestsents-märgitud nukleotiide, puhvrit ja Taq polümeraasi (hoida jääl!) vt lisa. ja www aadress: http://www.gelifesciences.co.jp/tech_support/manual/pdf/us81050pl_rev_c.pdf 75% isopropanool 99% formamiidi 5 mM EDTA-Na2 lahus. Töö käik:
kontsentratsioon? MgCl2 kontsentratsioon: (25/100)*8=2mM/µl dNTP kontsentratsioon: (2/100)*10=0,2 mM/µl Praimerite lõplik kontsentratsioon: (100/100)*5=5 mM/µl 4.5) PCR on eksponentsiaalne. Mitu DNA fragmenti on teoreetiliselt võimalik saada ühest DNA molekulist 5, 15, 30 tsükliga? Ühest DNA molekulist on võimalik saada 5 tsükliga 2 5 fragmenti, 15 tsükliga215 fragmenti ja 30 tsükliga 230 fragmente. 11 Töö nr 5: Rekombinantse DNA sekveneerimine ,,DYEnamic ET Terminator Cycle Sequencing Kit" abil. Lähteained: Rekombinantne, inserti sisaldav DNA (~100 ng/l) T7 RNA polümeraasi promootori praimer (100 ng/l) "Sequencing reagent premix" (Amersham), sisaldab fluorestsents-märgitud nukleotiide, puhvrit ja Taq polümeraasi (hoida jääl!) vt lisa. ja www aadress: http://www.gelifesciences.co.jp/tech_support/manual/pdf/us81050pl_rev_c.pdf 75% isopropanool 99% formamiidi 5 mM EDTA-Na2 lahus. Töö käik:
will bring government, academia, and the private sector together to identify the best applications as quickly as possible and expedite funding. "Innovative research in nanotechnology will impact a variety of critical areas in the future," says Alain E. Kaloyeros, Ph.D., vice president of CNSE. "In healthcare, for example, nanotech will be used to develop biochips for blood testing and DNA sequencing, smart chips for spinal cord injury and other physical challenges, chip-controlled patches for drug delivery and monitoring of bodily functions, and non-intrusive sensors for detection of disease, such as cancer cells." Other key sectors that will benefit are alternative renewable energy (environmentally friendly biofuels), solar cells and photovoltaics, and national defense. "The power of nanotechnology is rooted in its ability to impact multiple industries," says Kaloyeros
Handouts? Question Questions? Developing your talk / linking ideas Moving on Let's (now) move on to/turn to ... I now want to go on to ... If I could now turn to ... This leads/brings me/us to ... I'd now like to move on to/turn to ... So far we have looked at ... . Now I'd like to ... Turning to ... Now, what about ...? Next ... My next point is ... Let's now look at ... I'd like now to ... Let me now move on to ... Sequencing/Ordering Firstly ... secondly ... thirdly ... Then ... next ... finally/lastly... Let's start with ... Now we come to ... Let's leave that ... Let's get back to ... That covers ... Asking checkup questions Are you with me so far? Is everyone with me? Is that clear to everyone? Before I go on, are there any questions about ...? Using rhetorical questions What should we do? (pause) How much would it cost? (pause) Dramatic structures We have a revolutionary product
jarjestus) Nukleiinhappeline hubridiseerimine -tanu DNA, RNA molekulide voimele siduda vabasid Nhid on voimalik teataud NH-jarjestusega vabade margistatud DNAfragmentide abil avastada komplementaarse jarjestusega loike uuritavas DNA voi RNA molekulis. DNA kloonimine -uhe DNA fragmendi alusel on voimalik sunteesida sama fragmendi miljoneid koopiaid. DNA fragmendi nukleotiidide jarjestuse maaramine (sekveneerimine-ingl k. sequencing), mis voimaldab maaratleda geenide NH-lise koostise, nende tapse asukoha kromosoomis, aga ka geeni poolt kodeeritavate valkude aminohappelise koostise. Insenergeneetika -geenide DNA jarjestuse muutmine ja muudetud geenide voi uute geenide viimine rakkudesse ja organismi. Organismide geneetiline modifitseerimine. Restriktsiooni-ehk piiravad ensuumid 1970. a. avastati, et paljudel bakteritel on omadus lohustada bakterirakku tunginud voorast DNA-d (peamiselt bakterviiruseid) fragmentideks.
for explicit permissionfrom the primary station, to transfer anything. Acknowledged connectionless ("Type 3") provides acks, but For example, if the PPP link is configured to connect with IP, Advantages · Large packet size = low overheads = high efficiency · Batch Transfers · Auto receive · Asynchronous Response Mode (ARM): the secondary does not have to wait doesn't provide error recovery, sequencing or flow control. then the Internet Protocol Control Protocol (IPCP) will Disadvantages · Relatively susceptible to noisy lines · Not available in all comms packages Kermit: to receive explicitpermission from the primary to transfer any frames. · Connection-oriented ("Type 2") mode is similar to TCP. negotiate and configure the link to carry IP.
piirkonnas (iga ensüümi jaoks eri NH järjestus) (2) Nukleiinhappeline hübridiseerimine- tänu DNA, RNA molekulide vôimele siduda vabasid NHid on vôimalik tetaud NH-järjestusega vabade märgistatud DNA-fragmentide abil avastada komplementaarse järjestusega lôike uuritavas DNA vôi RNA molekulis. (3) DNA kloonimine- ühe DNA fragmendi alusel on vôimalik sünteesida sama fragmendi miljoneid koopiaid. (4) DNA fragmendi nukleotiidide järjestuse määramine (sekveneerimine- ingl k. sequencing), mis vôimaldab määratleda geenide NH-lise koostise, nende täpse asukoha kromosoomis, aga ka geeni poolt kodeeritavate valkude aminohappelise koostise. (5) Insenergeneetika- geenide DNA järjestuse muutmine ja muudetud geenide vôi uute geenide viimine rakkudesse ja organismi. Organismide geneetiline modifitseerimine. 3. Mis on restriktaasid? Restriktaasid (sait-spetsiifilised endonukleaasid) on ensüümid, mis lõikavad DNA-d spetsiifilise nukleotiidse järjestuse järgi. 4
Nüüd katseklaasis kasutades pöördtranskriptaasi sünteesime sellele mRNA'le ühe kaheahelalise DNA koopia. Seda koopiat kutsutakse sidiDNA'ks (?). Ja kõik jätkub endisel viisil. 05/11/09 Erinevad bakterid sünteesivad erinevaid restriktaase, erinevatel restriktaasidel on järjestusspetsiifika. Kui tahame kindlat lõiku DNA'st, siis valime sellise restriktaasi, mis lõikab katki sobivaid järjestusi. Kuidas me teame DNA nukleotiidset järjestust? Sequencing sekveneerimine. DNA nukleotiidse järjestuse väljaselgitamise meetodi mõtles välja F.Sanger. metoodika baseerub sellel, et Sanger võttis kasutusele nn modifitseeritud nukleotiidid. /ribonukleotiidis on nii 3' positsioonis ja 2' positsioonis on riboosi jäägi küljes OH rühmad, desoksüribonukleotiidis on hapnik puudu 2' positsioonist puudu. OH rühm on vajalik fosfodiester sidemete moodustamiseks./ Modifitseeritud nukleotiidil puudub OH rühm ja ahela polümeriseerimine sellest
5 kromosoomi, genoom 125 Mb. Umbes 25000 geeni. 35% geenidest unikaalsed, Lisaks nukleaarsele genoomile ka mitokondriaalne ja kloroplastide genoom, Geenid kompaktsed s.t. väikeste intronitega ja paiknevad genoomis lähestikku (keskmine vahemaa 4.6 kb). Mitmed geeniperekonnad paiknevad tandeemselt, Eksonid on palju rohkem G+C rikkad kui intronid (taimede omapära), Genoomis toimunud arvukalt duplikatsioone (polüploidiate teke). Mus musculus genoom: Lõpetatud aastal 2002. MGSC (Mouse Genome Sequencing Consortium) poolt 7x kaetud ja katab 96% genoomist (2,6 Gb), hiireliinil C57BL/6j. Avalik andmebaas, Celera hiire genoom 2001 a. Genoom 6x kaetud. Liin 129x1/SvJ,DBA/2J. Kommertsiaalne andmebaas, 2004 a. 90% Rattus norvegicus genoomist (2,75 Gb) RGSPC poolt. 8. Inimese genoomi projekt (ajalugu, eesmärgid, ELSI, strateegiad). 1956 a. esimene inimgenoomi füüsikaline kaart, 1977 a. Sanger: dideoksü-sekveneerimise metoodika, 1980 a. Botstein: geneetiline kaart RFLP-de alusel, 1981 a. Sanger:
tation–growth challenges) has been the first fermented sausages is fluorescence in situ to be applied and is still in use, despite its hybridization (FISH). In this technique, the drawbacks regarding reliability and accu- direct detection of a microorganism in a food racy, even at species level. The most fre- sample is achieved by using specific probes quently applied and most reliable technique that allow spatial distribution studies to take is sequencing of the 16S-rRNA gene. This place. technique has been applied either in combi- The microorganisms that are most fre- nation with other techniques, such as SDS- quently encountered are Lactobacillus curva- PAGE of whole cell proteins, RAPD-PCR, tus, Lb. plantarum, Lb. sakei, Staphylococcus or PFGE, or directly to the isolated microor- carnosus, St. saprophyticus, and St. xylosus ganism. Although this approach provides (Table 9.1)
solution to create a molecular sieve also known as replaceable physical gel. This allows analytes having similar chargetomass ratios to be resolved by size. This technique is 16 commonly employed in SDSGel molecular weight analysis of proteins and the sizing of applications of DNA sequencing and genotyping. Capillary Isoelectric Focusing (CIEF) CIEF allows amphoteric molecules, such as proteins, to be separated by electrophoresis in a pH gradient generated between the cathode and anode. A solute will migrate to a point where its net charge is zero. At the solutes isoelectric point (pI), migration stops and the sample is focused into a tight zone
usually do not agree with general project goals. This is the opposite of what the main contractor managers are trying to achieve, namely, the best value and results for the customer. Control operation, based on thermostat theory, is to monitor if a project is within cost and time limits. Action is taken only when delays in work appear. The techniques used to improve the situation often consist of speeding up sub-contractors, bringing in additional workers or re-sequencing jobs so that more important ones are finished first. Thus, managers are busy fixing the tasks that were not delivered on time, and this leaves them less time for planning upcoming tasks. This leads to the following project results: low productivity, poor safety, inferior working conditions, and insufficient quality. Winch has stated the following causes for poor project management more succinctly, but they are basically the same as discussed above:
Samuti on tarvis kontrollmehhanisme ja vigade parandust, mis nõuab samuti täiendavat energiat. 41.(136) Ligikaudu mitu erinevat valku on inimeses? 4 a) 500 b) 5000 c) 50000 siit võtaks C(50 000), kuid kui netist vaadata siis hinnatakse seda arvu 8590 tuhande peale. Ühel Väljamäe slaidil on kirjas: Homo sapiens 30 000 100 000 valku. After the completion of human genome sequencing project in the year 2000, the consensus has been that there are between 40,000 to 100,000 genes in the human genome. Consensus also exists that there are two to three fold more number of proteins in human proteome as compared to the total of number of genes in the human genome. However, given this staggering number of proteins that are known to exist in human body, there are only about 500 proteins so far that are known to be the targets of all the
Genoomi kaardistamiseks kulus 13 aastat. Järjestuse analüüs paljastas, et inimesel on umbes 20 000-25 000 geeni. Inimese terve nukleotiidse järjestuse teadmine annab võimaluse leida haiguse tekkes osalevaid geene ning õppida paremini tundma haiguste tekkemehhanisme. Samuti aitab see teadmine tulevikus võimaldada personaalset meditsiini ehk genotüübist sõltuvat ravistrateegiat. 57. Mis on massiivne paralleelne sekveneerimine (massive parallel sequencing) NGSi ehk massiivse paralleelse sekveneerimise esimeseks etapiks on genoomse DNA fragmenteerimine väiksemateks lõikudeks, millest seejärel koostatakse raamatukogu. Raamatukogu lõigud kantakse kandjale, kus toimub DNA fragmentide seondumine kindlate proovidega ja kaksikahela denaturatsioon üheahelaliseks. Edasiselt sünteesitakse uuritavale üheahelalisele DNA järjestusele vastasahel. Iga üksiku nukleotiidi lülitamisel
Digital Sound Processor + Directory Synchronization Protocol [Lotus] DSPT Display Station Pass-Thru [IBM] DSQD Double Sided, Quad Density (diskette) DSR Data Set Ready + Device Status Register + Device Status Report DSS Decision Support System + Digital Signature Standard + Digital Spread Spectrum + Direct Station Selector DSSA Domain Specific Software Architecture DSSI Digital Standard Systems Interconnect [DEC] DSSS Direct-Sequencing Spread Spectrum DSSSL Document Style Semantics and Specifications Language DSTN Double Supertwisted Nematic DSU Data Service/Switching Unit + Digital Service Unit DSVD Digital Simultaneous Voice and Data DSW Data Status Word + Device Status Word DSX Digital Signals Cross-Connect D2T2 Dye Diffusion Thermal Transfer (printing) DTA Digital Terminal Adapter + Disk Transfer Area .DTA Data (file name extension) DTC Desktop Conferencing
annaabi.ee 
