Questions, answers and sum of main ideas for G&G test 1
Questions, answers and sum of main ideas for G&G test 1 PSS8653
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STUDY GUIDE – GENOMICS AND GENOMES – TEST 1 – ANSWERS PART 1: BASIC CONCEPTS 1) Genome definition rely on the type of organism; organelle or virus it is associated. Mitochondria and viruses, even though it is questionable if they are living creatures, they still have genome. Generally, genome is a minimum amount of DNA sequence that characterize an organism/organelle/virus. 2) Genomicsisthestudyofallgenes,ofthegenome.Methodsusedincluderobotics,advancedcomputational biology and sequencing. 3) While genetics look for specific genes, “fishing a specific target”, genomics collect all genes possible and then focus in specific regions of the genome. 4) Even though some things do not have genome, they are still important contaminants of samples that contain genome, except fire. Contain genome No genome Eukaryote Dirty Chloroplast Rocks Mitochondria Air Prokaryote Fire Viruses Water 5) The biggest failure of scientific education is discriminate what is true science and what is not. Science is a method of study that rely on natural events/processes to test hypothesis and answer a question (biochemistry, biology). The roots of science are getting a hypothesis, testing the hypothesis, observing data, analyzing responses to then get into a conclusion. 6) Non-scientific believes are mainly religion and pseudoscience. Religion is based on non-scientific methods, myths, stories. Religion is usually based on supernatural events. However, pseudoscience uses some scientific jargon do get credence from people. One example of pseudoscience is creationism, which was used by Hittler to convince lots of people to make his own believes. 7) Evolution is the changing of the genome over time, over thousands and thousands of generations due to selection, mating choice or even migration and geographical aspects that ends up with speciation events. Genomics is a big field that held researches involving the evolution of species. Evolution was first described by Darwin (1850). 8) The central dogma is how the genetic material ends up on producing proteins and giving a phenotypic characteristic to an individual. In this case: DNA can replicate itself by DNA polymerase or can be transcribed in RNA by RNA polymerase. To produce proteins messenger RNA is transcribed and then translated by ribosome complex. Translation is based on the genetic code, in which every 3 base pairs represents a codon. Each codon represents either an amino acid or a signal for start/ stop codon. Start codon often AUG while stop codon usually UAA, UAG or UGA. Central dogma was first described by Crick and Watson. 9) Nucleic acids are classified as ribonucleic acid and deoxyribonucleic acid. The difference if that ribonucleic acids have an extra hydroxyl group at the2C of the sugar pentose, while deoxyribonucleic acid has it at only the3. Ribonucleic acids will form the RNA sequence chain, while deoxyribonucleic acid will form the DNA sequence chain. 10)The nucleotide structure involves: (1) phosphate group – usually triphosphate group (α,β,γ), (2) nitrogen base, which can be uracil, guanine, thymine, adenine, cytosine (3) sugar pentose chain. The process in which one nucleotide binds to another is called phosphodiester bond. In this case the hydroxyl group from C re3cts to the other hydroxyl group at the C , re5easing one molecule of water and a diphosphate group. 11)DNA molecule characteristics include: - Double helix structure made by H bonds and gives DNA stability - Complementary antiparallel double stranded molecule - Backbone phosphate and sugar pentose stay outer the structure due their negatively charged molecules, while bases stay inner - Deoxynucleic acid – less one OH group - Base pairing: A-T (two H bond) and C-G (three H bond), turn CG more stable and resistant - One strand ends with 5’ phosphate group and 3’ hydroxyl group 12)Replication Information: - It is based on the natural process of denaturation and renaturation of the DNA molecule. Under high temperature (95°C) and extreme pH (alkaline pH), DNA molecule can separate both strands by undoing the H bonds (denaturation). However, once the normal conditions come back, DNA molecular has the behave of getting together to its complementary strand (renaturation). Many molecular techniques based on this process to happen, such as PCR, blotting, microarray. - Direction: 5’ to 3’ – always getting new nucleotide to the 3’ hydroxyl group - Molecules involved: primer (short 5-10 bases, complementary to a particular location at DNA molecule), DNA polymerase, dNTPs (A,T,G,C). - Initiation sites, where the process of DNA replication begins, are usually A-T rich, because it is easier sites to DNA double stranded get separated by helicase or high temperature. - Each initial strands are used as template to produce two new daughter strands. So it is called semi-conservative process. - It is bidirectional process. -Events: topoisomerase reduce supercoil > helicase recognizes initiation sites and open the dsDNA and recruit DNApolymerase>aprimerisproducedandthendestroyedbyaprimaseat thereplicationfork>atthereplication fork DNA polymerase starts adding to the 3’ primer new complementary nucleotides in a continuous form > it happens at the leading strand which has a 5’ to 3’. At the other strand that has a 3’ to 5’ end orientation, called lagging strand, the strand is processed by more than one DNA polymerase in small fragments called Okazaki fragments and are put together to form the daughter strand. 13)RNA main ideas: - single stranded polynucleotide chain, hydroxyl group at C an2 C , ma3e by NTPs (U,G,C,A). - form a secondary structure folding on itself - no double helix, so it turns it to be less stable when compared to DNA - formed by using a DNA template (antisense strand) and RNA polymerase -ribozymes areRNAs that have enzymatic activity, oneofthemis responsiblefor the formation of protein chain, called peptidyl transferase ribozyme, in a reaction called peptide bond. - because all RNA functionality and organisms that use RNA as method to produce their DNA, it is hypothesized that RNA was the first genomic structure that was replaced by DNA during evolution. mRNA tRNA rRNA Regulatory RNA -Template for protein -Transports amino acids -most common RNA - responsible mainly for synthesis till the ribosome complex (count to 80% of all controlling gene -Eukaryote is synthesized to be added in the protein RNAs) expression on nucleus, modified and chain -form a complex -includes micro RNA, transported to the -Formed by 3 bases pairs responsible for non-coding RNA, cytoplasm where it is (anticodon) region that translation of mRNA to interference RNA translated must be complementary protein chain. It is part of -Prokaryote all process to the codon region at the ribosome structure occurs at the cytoplasm mRNA so AA can be -transcribe from parts of and no post translational inserted the genes called rDNA, modification occurs -Each AA has its specific found in repeats on the -carry the genetic tRNA, so at least 20 genome material code types exist -often called transcripts -Methyl tRNA is always -classified as mature or the first one to be pre-mRNA. brought at the translational site -most conserved across species, have a clover leaf 2D and L-shaped 3D structure well known. 14)Transcription information: - Transcription allows the cells to (1) develop and growth (2) differentiate from other cells, having its specific morphology (3) develop their function. Because it is by transcription that proteins are made afterwards and genes can be regulated properly. Different gene expression allows cells from same DNA material have different morphology and function. - Direction 5’ to 3’ - Molecules involved: transcriptional factors, enhancer or initiation factors, promoter region, RNA polymerase, NTPs (U,G,C,A). RNA polymerase does not essentially need a primer. Thymine is substituted by uracil. - DNA strand used as template is called antisense, so the transcribed RNA will be equal to the sense strand of the DNA. It is not always the same strand that is used as template. It chances from different genes and individuals. - Events: transcriptional factors recognize the promoter region on DNA upstream the sequence> recruit RNA polymerase> transcription initiation complex > activator factors recognize enhancer regions and once they attach, some transcriptional factors are released so RNA polymerase is free to move and start transcription > RNA polymeraseattach NTPsfroma3’andonceit finishestranscriptionRNAisreleased>duringtranscriptionmRNA have post transcriptional modifications in Eukaryote - Transcriptional factors and enhancer/silence sites are methods in which transcription is regulated. -Post transcriptional modifications include: (1) methylation of 5’ cap (2) poly A tail at 3’ (3) splicing of intromic regions. Prokaryote do not undergo splicing on their mRNA. -pre-mRNA is the just finished RNA, which haven’t undergone post transcriptional modifications. While mature mRNA is the one that had all post transcriptional modifications done. Usuallygenomics studythe mature mRNA. -cDNA is a type of DNA sequence that was originallya RNA that was reverse transcribed to DNA. It is how most of retroviruses work. 15)Amino acid information: - total count for 20 types of amino acids (AA) - classification of AA: CLASSIFICATION CHARACTERISTIC Acidic Negative charged Basic Positive charged Non-polar No charge Uncharged Polar Each end has a different charge Hydrophobic Non water association. Usually formed by non-polar AA. Stays at the inner sites of a protein structure/chain. Hydrophilic Stays outer side of a protein structure. Usually formed by acidic, basic and uncharged polar AA. - the amino acid chain is formed by peptide bond which is catalyzed by the peptidyl transferase ribosome. In this process, at normal physiological conditions the amino acids are ionized facilitating the reaction between the amino group from one amino acid to the carboxyl group from other amino acid. This reaction releases water molecule. OH from the carboxyl group reacts to the H from amino group. -AA structure is mainly: (1) amino group NH ,3(2) carboxyl group COOH, (3) central carbon Cα and (4) the side chain or R group that differentiates each type of AA. 16)Translation information: - It is the process in which the mRNA molecule is read codon by codon and a respective amino acid associated specifically to each codon is introduced in an AA chain (protein). - There are only a few differences between eukaryote and prokaryote translation. It is pretty much conserved across species. - Ribosome is a complex formed by proteins and rRNAs that is responsible for the translational process. It is formed by small and large subunit. The small subunit is where the mRNA passes through and is hold for its codon recognition, while the large subunit is where tRNA get inside and if anticodon from tRNA and codon from mRNA is a match, the AA is released to the form the chain and the tRNA is released. If no match happens, tRNA is released with its AA. There are 3 main sites at the large subunit: A = where the tRNA gets in, P = where AA is gotten to form the protein chain and E= where tRNA is released. - Events: small subunit of ribosome recognizes the mRNA > recruit large subunit > ribosome complex formation > first tRNA methylgets in to form the protein chain > matching codon anticodon more AA are joined to the chain > ribosome reads till the end and protein is released at the end 17)Definitions: - Gene: DNA sequence often responsible for controlling a discrete hereditary characteristic on an individual. It often corresponds to a single protein or RNA. - Locus: specific region on the chromosome where a gene is located. - Allele: one of a set of alternative forms that a gene can have. 18)Recombination can either occur by exchanging or replacing a portion of the genome. It requires a minimum homology to happens. PART 2: MOLECULAR CLONING 1) Restriction enzymes main ideas: - it was first described in bacteria that use these enzymes to protect themselves against foreign DNA that tries to invade the cell, such as viruses. -restriction enzymes are enzymesthat cut the DNAbackbone on both sides, originating a break on the DNA sequence. It targets specific palindrome sequences, which are short sequences that can be read back and forward as same sequence. For example: AGCGA. - resulting cut fragments can be classified as (1) blunt ended strands – which are symmetric cut of the DNA or (2) sticky ended strands – which are asymmetric and can be (2a) 3’ sticky end cut, in which the overhanging part is at the 5’ and at the 3’ we have the smallest cut piece (2b) 5’ sticky end cut that has the 3’ overhanging strand. - bacteria protect itself from restriction enzyme by having a non-methylated DNA. 2) The main principle of molecular cloning is getting two DNAs, cutting them with same restriction enzyme (RE) that produce sticky ends and putting them together so they will recombine and form a singleDNAsequence. This procedureofteninvolves E.coli orotherbacteria,plasmids, atarget (insert) DNA and a RE that produce sticky ends, such as EcoR1 or HindIII. Events: two tubes, one with plasmids and other with target DNA > RE added to both tubes > mix the open plasmid with the small strand of DNA target > by complementary bases on the sticky ends insertion occurs > plasmids are inserted on bacteria by either electroporation or the bacteria take up by themselves > growth bacterial colonies > genetic markers can be used to select only the bacteria that presents the recombined plasmid. 3) The most common way of selection of bacterial colony is by α-complementation. In this method the intact plasmid had an intact LacZ gene. The inserted foreign DNA breaks down the LacZ gene, so the bacteria with recombined plasmid cannot efficiently metabolize the lactose. By using specific media for culturing the cells, recombined bacteria will have white color while not recombined bacteria(due to no absorption of plasmid orhavinganintact plasmid)will havebluecolor. So, white colonies are selected. 4) Constituents of a clone: - Insert: the target foreign DNA that will be inserted on the vector. Called DNA clones. - Vector: is a DNA sequence in which will receive the foreign DNA. Often is a plasmid, but viruses can also be used as same purposes. - Host cell: cell in which the vector will be transferred and will produce copies of the target DNA or its product (protein). Vector can be transferred to the host cell by using competent cells (absorption naturally) or doing electroporation (shock treatment on the cells that make their membrane permeable). 5) Advantages of inserting a clone into bacteria: a. low cost and fast method, easy to assay b. E. coli can divide once every 20 minutes in ideal conditions. At the end of 10h can have billions of bacteria. c. DNA clones will be amplified without using PCR methods. 6) Main conditions to E.coli to growth: enough space on a plaque, no competition, fresh media and 37°C. 7) Plasmid is an extrachromosomal DNA. Often circular and double stranded, that can replicate itself whenever it wants. It does not require the cell to be on replication. Its main features include: a. Genetic Marker: it is a gene that allows the identification of the bacteria that had up taken the recombined plasmid, such as LacZ gene. It is related to the polycloning sites, because once an insert gets into the plasmid, it inactivates the marker causing changing on phenotype. b. Antibiotic resistant gene: gives advantage for growing bacteria, such as ampicillin resistance. You can also select bacteria by growing them in a media with antibiotic, so you know that the ones that are replicating have the plasmid. c. Polycloning sites: region formed by short sequences in which RE will act and the DNA will be inserted, such as the β-galacto site. Only a specific region has these sites on the plasmid. d. Partitioning genes: form genes that clock the entrance of other plasmids inside the bacteria, so only one plasmid per bacterium is found. It is naturally found in bacteria. 8) Plasmid is not very often found in bacteria. Usually few bacteria have plasmids. 9) DNA cloning libraries are the DNA products from a cloning. They can be classified as: a. Expression clone libraries: in which the plasmids also contain genes that allow the transcription and translation of the insert DNA. Vector needs to have promoter and initiation regions. So proteins can be analyzed and kept. This one is pretty much used for pharmacological purposes, such as production of human insulin. The gene responsible for producing insulin is cloned and the protein formedbybacteria. Beforeit was collectedfrom pigs andwouldcausereactions onhumans and take lots of pigs to obtain insulin. b. Genomic clone libraries: formed by the target gene that was inserted. c. cDNA clone libraries: formed by mRNA that were reverse transcribed to cDNA and inserted into a clone. 10)The main methods of picking up colonies of bacteria are (1) by hand using tooth pick or (2) robots (select round, white, lonely colonies). Procedures after collecting colonies include: - Transferring colony to a new 384 well plate - Overnight growing conditions (inoculation) - Identifying the plate (bar codes) - Replicating the plates (usually more 3 plates) - Refrigeration -80°C 11)Main methods of plasmid/ DNA isolation include: - Electrophoresis gel - Pulsed-field electrophoresis - Blotting techniques - PCR - FISH 1. ElectrophoresisGel:useagelofagaroseoracrylamide.Thesegelshave porous that allows the DNA to pass through. The gel is placed on an electro field container that has two fields (+) and (-). Over the gel a solution buffer is placed. Because the DNA backbone is negatively charged, it migrates on the gel to (+) cathode. So, DNA is placed on small wells close to the (-) anode and migrates all over the gel trying to get closer to the (+) cathode. Larger fragments of DNA run slowly through the gel, so often stay more close to the initial well. Smaller fragments run faster and so go farther away from the initial point. So rate of migration is inverse proportional from the size of the DNA. Advantage: it is relatively cheap (mainly the agarose), easy and fast. Used when the DNA fragments are not much large. 2. Pulsed-field electrophoresis: it works basically just like the first one. However, instead of 2 fields (+) and (-), it is a 6 sided field in which the gel is placed in the middle, also using a buffer. The idea is the with increasing fields larger fragments can run through the gel without getting stuck. Each well can have more than 100 fragments with thousands bp. 3. Blotting techniques: basically it is a post electrophoresis method to obtain DNA or bacterial colony. Once you have your gel with the separated DNA fragments, a nylon ornitrocellulosefilteris placedoverthegel and over the filter a type of absorbent sponge. Under the gel, the buffer solution is kept. So, the buffer runs into the absorbent sponge by capillarity, carrying together the DNA fragments. Due to the charge of the filter, DNA is retained there. Type of blotting techniques include: - Western blotting: used to separate proteins -Southern blotting: used to separate DNA -Northern blotting: used to separate RNA -Colony blotting: used to separate bacterial colonies. After getting the colonies on the nitrocellulose or nylon filter, 0.5N NaOH is used to break the membrane of the bacteria and turn dsDNA into ssDNA. Proteases and other buffers are used to only leave intact DNA. All of them you can use a final target labeled (probe) that will recognize specifically the DNA fragments you want and fluoresce when binding to it. 4. PCR methods: it is an exponential amplification of DNA fragment. basically you will have primers designed for the specific DNA fragment you want. Different thermal cycles will be performed so you can have amplification of your target DNA: - 95C: DNA denaturation, open the dsDNA in ssDNA - 55-60C: primer annealing - 72C: DNA polymerase (Taq poly) extend your DNA sequence As replication, it requires: (1) DNA polymerase (2) dNTPs (3) primer complementary to the vector flanking region. Most used DNA polymerase is Taq polymerase, isolated from Thermus aquaticus, a thermophilic bacterium. So the polymerase is resistant to high temperatures. n At the end of first cycle you have 2 copies of DNA from the first DNA. At the end of ‘n’ cycles you have 2 copies. Usually 30 cycles are performed in a PCR. Advantage: more than isolate, more sequences are obtained. Disadvantage: size. Really long sequences won’t work because polymerase might fall at some point. 5. FISH: fluorescent in situ hybridization. It is a means by which genes and/or repeat sequences are visualized in the chromosome. This method is used to help recognizing the DNA sequences that you want. Basically you have your DNA sequence or chromosome and you insert it in a glass. The glass is heat up and chemicals are added to denature the DNA and turn it into ssDNA. Complementary specific probes bond to a hapten recognize its specific region on DNA sequence and hybridizes. After hybridization, another molecule is added to the glass. This has a fluorescent characteristic and is highly specific to the hapten. Once this molecule binds to the hapten, light is produced and captured by the machine. - hapten is an inert small molecule that does not affect hybridization procedure or biological process, such as biotin. - fluorescent tag specific for the hapten can be either another molecule such as streptavidin (highly specific to biotin) or an antibody. Uses in studies of (1) genetic recombination (2) relationship between genes (3) chromosome structure. 12)Molecular genetic markers information: - Definition: short sequence of DNA characterized by polymorphisms (multiple forms) - To be useful it needs to be easy to assay and distinguish from different forms. - It is used for breeding experiments, genetic diseases, evolutionary studies - Molecular map definition: is the association between molecular markers and its location at the chromosome. - Molecular recombination marker is based on the rate of recombination and the present of molecular markers. -Main types of molecular markers include: (1) RFLP (2) PCR based marker (3) Linkage marker (4) microsatellites (5) SNPs * RFLP: restriction fragments length polymorphisms. It is a technique in which DNA is cut in specific regionsbyrestrictionenzymesandthepatternsoffragmentsareanalyzedintoagel.Mainusesinclude: paternity test, diagnostic of genetic diseases, DNA profiling. It is the test used for diagnosis of sickle cell anemia, in which sick people presents a shorter βglobin gene compared with normal individuals. * PCR based marker:usuallyuses semi-randomamplificationofDNA. IncludestheAFLP (amplified fragment length polymorphism) and RAPD (randomly amplified polymorphism DNA) *SNPs is probably the most efficient and accurate. However, it is also the most expensive once it involves sequencing. It basically localizes each polymorphism in a DNA sequence. * Microsatellitesareshort sequences on DNAthat arerepetitive. It canbe dinucleotiderepeats (AT) , n or trinucleotides repeats (AGC) and so on. They have been studied and as involving not only on diseases but also social behaviors. *Linkage marker shows the association between recombination and the markers. So how often markers appear together means the possible association in recombination events. Gives an idea of distance of markers within a chromosome. PART 3: SEQUENCING 1) Sequencing is the method is which determines the order of each base pair of a DNA molecule. It started around 1970s and have been still improving. 2) Two mains sequencing methods were created almost at same time. (1) Maxam-Gilbert sequencing or Chemical sequencing based only on chemical steps that end up differentiating each base pair from a sequence (2) SangerSequencingor Chain Terminatorsequencingthat was basedonbiochemical methods, included biological events and elements such as DNA polymerase, replication methods. 3) About Chemical Sequencing: - All other answers are present in a table comparing chain terminator and chemical sequencing as follow. For the gel, remember that it can only be a G is it appears on both columns (G and G+A), otherwise it is a A. For a base be C it only happens if it appears on both columns (C and C+T). 32 Sometimes, the first G ligates to 5’P is not shown in the gel, so just keeptrackingafter theP pGfragment. It is becausemost of the time the P is bond to a guanine. 4) The larger structural bases are purines (adenine and guanine), which have another pentose like sugar. While, pyrimidines (thymine and cytosine) have the shorter structure, no extra pentose. 5) About Chain Terminator Sequencing: - Sanger created it, but his first award was finding the sequence of insulin protein by protein methods. Method Chemical Sequencing Chain Terminator Sequencing Who created it Maxam & Gilbert Sanger – first method that sequenced the whole genome from an organism (phage) – he got his second Nobel prize with that Uses / famous method No, almost not used. Yes, it was modified and is still Only for specialized experiments. commonly used nowadays. Method Low cost. was modified so no agaroses gel is needed nor 4 different reactions. Main Characteristic Onlychemical substances areusedto ddNTPs – modified base pairs that define each base from a molecule do not present the hydroxyl group at the 3’ end, so whenever DNA polymerase try to elongate the sequence it stops and only a small fragment is formed Initial Steps *DNA sequence is break into * same initial steps from chemical fragments sequencing *alkaline phosphatase removes 5’ phosphate *ATP +nucleotidekinaseareusedto insert an radiolabeled phosphate group at 5’ = P2 Denaturation Chemical denaturation of DNA Temperature denaturation Processing sequencing *ssDNA sample is divided into 4 *ssDNA sample is divided into 4 tubes: tubes: 1. dimethyl sulfate = take out purines - each tube will contain DNA (depurination), more specific for G polymerase, dNTPs, primers and the over A target DNA 2. formic acid= take out purines - the difference is that in each tube (depurination), more specific for A one type of base will be substitute by over G ddNTPs, as follow: 3. Low hydrazine = take out 1. ddATP pyrimidines (depyrimination), C and 2. ddTTP T are equally chosen 3.ddGTP 4. Hydrazine + 0.5N NaCl = take out 4.ddCTP pyrimidines, more specific for C over T Breaking down in fragments For purines (tube 1 and 2), NAOH is DNA polymerase does during the included to break the phosphate elongation process backbone where the base was firstly removed For pyrimidines (tube 3 and 4), piperidine is added to break phosphate backbone where base was removed Separation of fragments and Electrophoresis gel – separate larger *same as Chemical, however each analysis fragments till small fragments. By column in the gel will be specific for each tube is possible to tell which a type of base base pair it is and by the size of fragment where the base is located on the sequence. The main differences between a deoxynucleotide and a di- deoxynucleotide. Reminder: ribonucleotide presents 2 hydroxyl groups, at C 2 and C 3 Deoxynucleotide presents only one hydroxyl group at C 3 while dideoxynucleotide does not have hydroxyl group at all. 6) AZT, known azidothymidine, is another modified base, more specifically modified thymine base, in which instead of hydroxyl group at 3’, this molecule presents a nitrogen molecule at 3’. At the same way as the ddNTP said before, DNA polymerase cannot elongate a DNA chain during replication if this molecule is taken up. AZT is used for HIV treatment because the virus prefers using AZT during elongation instead of normal thymine. However, the DNA polymerase stop replication and the virus cannot be replicated. Because human cells do not have affinity for AZT, it is not taken up during replication so low side effects are seen for the use of this drug (increased safety). Once AZT is taken, the human cells convert it to triphosphate base. 7) About Dye Termination sequencing: -Improvement of Sanger sequencing by: (1) each ddNTP is radiolabeled with a different color, so all reactions can be performed at only one tube (2) no need for phosphate radiolabeling, no P 32(3) uses capillary gel (4) form an electropherogram. - Originally acrylamide gel was used to separate the fragments. Nowadays, fragments can be visualized by capillary gel, which a machine perform a gel in a tiny capillary tube and a laser read each color from each fragment that is separated. -After the laser reads the signals, it gives an electropherogram in which each color and peak represents a specific base. If double peak is shown or N appears, it means that the machine could not tell which base it was. -Disadvantage: radioactivity labeling is biohazardous. 8) About Cycle sequencing: -Main improvement/difference: it is a PCR-based method using a thermocycler. -Main idea: you can use a thermocycler to perform amplification of your sequence and by using ddNTPs with different tags you can produce fragments that will be identified afterwards in a gel. -Advantages: (1) less DNA template required (2) amplify your DNA template during sequencing so increase accuracy, high quality reads (3) once kits got commercially available, it is low cost (4) no chemical denaturation is used (5) even difficult templates can be performed. -The usual DNA template comes from an insert from plasmid. So, the primers are complementary to the polycroning sites from the plasmids. -Process 384 samples at time/run. -Reads up to 700-1,000 bp (limit) -Disadvantages include: (1) limited length of DNA template (2) expensive to perform in small scale -Capillary gel are smaller tubes in which the laser read each fluorescent tag that passes through it. It’s twice faster than slab gel and minimum human work. 9) About Paired End sequencing: -Also called 2 pass sequencing or Bidirectional sequencing -Basically it is used for larger DNA sequences length, often coming from a plasmid. So, the primers will be 2, each one will be complementaryto a different end and different strand from a polycloningsite region from a plasmid. The amplification and sequencing reaction is performed in two different tubes, each one with one type of primer. While one tube amplifies one strand that originally was the 5’-3’, the other one will amplify the strand that was originally 3’-5’. After amplification that follows a sanger sequencing method (more likely cycle sequencing), the strands are read by capillary gel and the final sequences can be either paired end sequences or form a consensus sequence. -Paired end sequences are cases in which the original template was more than 1,400bp long so when the sequencing is complete and analyzed both strands it will have a gap within both strands. The gap or missingpartcanbefoundoutbyperformingatargetPCRtofindthatregion orcantrytofindinareference genome. -Consensus sequence is formed when both strands give a complementary overlapping region that allows to write the whole sequence from 5’-3’. Usually the sense strand is the one chosen for representing the sequencing. If you don’t know either which strand is the sense, one is chosen at random written from 5’- 3’ direction. 10)About Next Generation sequencing: -The main differences/improvements are: (1) sequencing results are given at same time that amplification or sequence is being read (2) use polonies to amplify reads quality and accuracy (3) not based on electrophoresis. -It is based on polonies. Polonyis a junction of colonyand polymerase, which means a group of sequences extended by DNA polymerase forming a ‘colony of sequences’ that have similar sequences. The advantage is that as more molecules you have giving same information about a DNA fragment, more accuracy and more efficiently the machine you capture the signal. So, polony is group of million copies of same DNA template. Disadvantage: (1) expensive (2) slow down sequencing process. -Rely on the technique in which PCR is used to amplify millions of sequences attached to beads or to a slide embedded with acrylamide. -Subtract is a glass slide or 384-well plate that allows fixation of the DNA template so it fixed in a place and grouped together by its similarities. -Reads are the products of sequencing, usually short fragments of the original DNA template. They are put together to form the sequence of whole DNA template. -DNA libraries (different from DNA cloning libraries), are all products produced by sequence process. Include all reads. -In sum this method includes other methods that differ basically as followed: Method Main key 454 pyrosequencing Luciferase fluorescence when combined with diphosphates generated during nucleotide insertion Illumina Use a reversible blocked base at 3’OH. Clustering sequencing, many methods as bridge amplification, washing steps, indexing, allows high quality reads Solid sequencing Dibase probe are ligated to the complementary strand of DNA. Instead of replication, ligation is the method used. So at each time 2 bases are inserted in the growing chain instead of one base at time. Each pair of bases have its own fluorescence. SMRT / Pacific Biosciences Single molecule sequencing, where each molecule goes to a single 20 zeptoliter well to be amplified (ZMG). Laser passes through tiny hole to read the fluorescent tags emitted from each type of base. Bases are labeled at the 5’phosphate group that is released during insertion. Ion Torrent Semi-conductive chip that presents wells in which each well receive a bead containing a single molecular fragment of DNA. As nucleotides are inserted + during replication, H are released, pH changing is recorded as changing voltage of the chip. One base is given at each time. No fluorescence is used. Nanopore sequencing No amplification, no primers, no polymerase, no chemical reaction. Membrane with a tiny pore in each one strand of DNA will pass through once at time and differentiation of each base structure captured by a potential changes the membrane voltage and is registered by the machine. 11)About 454 pyrosequencing: -Created from a group called 454 and now it owned by Roche. -no cloning involved. -This method is based on chemiluminescence. In which light is emitted and registered. In this method, light is produced by luciferase that recognizes diphosphate groups released from phosphodiester bond, binds to it and reaction release light. So whenever a base is incorporate to a growing sequence, light is produced. -700Mb/run/23h, reads have ~1,000 bp. Around 1,000,000 reads/run. -Scheme (better scheme attached at end): DNA sequence > break into fragments> two adaptors > select sequences with both adaptors, one in each end> mix with solution containing beads > one sequence will attach perbead >oneadaptoris complementaryto shortstrandonthebead >oil solution = macroreactor> amplification occurs > each bead contains millions of copies from same original fragment > transfer bead to well > well will be fulfilled with a solution containing one type of dNTP at time > when polymerase incorporate a base, luciferase reaction occurs and light is emitted > machine compute the signal from each bead -Proportion of DNA that efficiently binds to a bead: 1 out of 3 -Paired End sequencing in 454: attach to DNA fragments a biotinylated harpin molecule at each end > cleaves part of this molecule by exonuclease > leads to circulation of the DNA > nebulization opens the DNA to get straight again and so biotin is kept then in the middle of the sequence > adaptors A and B are attached > sequences with both adaptors are selected by streptavidin -Disadvantage: time consuming, too much processes and it is complicated. -Not much used nowadays. Almost extinct. 12)About Illumina sequencing: -Also called clustering sequencing = Reversible chain terminator -Main differences compared to 454 sequencing: (1) based on Sanger sequencing in which you have radiolabeled bases (2) no beads are used, instead a flow cell (2) no luciferase (3) bases are reversible block at 3’OH for short time so machine can record the base that was incorporated in each cluster (4) based on paired ended sequencing -flow cell is a glass containing lanes. Each lane has two types of oligonucleotide sequences that will be complementary to one of the adaptors in the template DNA. -Scheme: DNA sequence > fragments of DNA > DNA poly and dNTPS with DNA will generate an A overhand > adaptors A and B attached > select sequences > apply sequences in glass > fixation > first amplification > denaturation > first product washed away > bridge amplification (function of increase number of sequences) > denaturation > cleaves and wash away reverse strands > initiate sequencing/amplification of forward strands > each base that is incorporate has a different fluorescence and is blocked from keep incorporation until machine register that base > denaturation > wash away first sequenced product > bridge amplification > same steps are repeated to the reverse strand. -Number of cycles is equal to length of sequence -Each read ~300bp (at first was only 25-50bp) -Indexing is the process in which more than one sample is processed, sequenced together. It is two ways to do it: (1) getting samples that are really different in sequence, no homology (2) bar coding. Bar coding is a process in which a short sequence of nucleotides is inserted on the template before the adaptors so after sequencing lots of samples you can separate them by the bar coding that is unique for each sample. It is a way to save money and have more results in one process. -Main idea: paired ending sequencing. A DNA template is sequenced twice, reverse and forward strand. -Advantages: (1) high quality reads, high accuracy (2) easy preparation (3) easy paired end sequencing. -Disadvantage: (1) length of sequence can limit the process (2) major issue is asynchrony, which means that as longeras asequenceis greateris theprobabilityofonesequencedon’t get anucleotideincorporated causing a differentiation on the produced length of the read. Over time it gets worse. HiSeq 4000 MiSeq NextSeq 1,300 Gb 13.2 Gb 100 Gb 2x150bp 2x300bp 2x150bp 3.5 days/ run 56h/run 29h/ run 13)About SMRT sequencing: -Single molecule real time sequencing -Owned by Pacific Biosciences – more close to the method of 454 sequencing -Big issues in the beginning. Full of empty promises. No prove that the sequence was accurate. Laser was destroying DNA polymerase over time. -Solved this issue by (1) repairing ends (2) purification process (3) DNA fragmentation (4) ligate adaptors. Sequel is the last version. -New about this method: (1) uses a tiny well in a chip with 20 zeptoliter (10 ) in which one DNA polymerase stays (2) laser passes through the hole and read the fluorescent emitted when a base is incorporated (3) bases have specific radiolabeled color tagged on 5’phosphate group instead of nitrogen group = phospholinked nucleotides -Comparing with Illumina, the rate of error is bigger once a single molecule is sequenced instead of millions. However, longer molecules can be sequenced with a length 20Kb! It is the longest read from amplification method! -Problem with managing long sequences is that it is difficult to process without breaking the molecule. However, it is easier to assemble after sequencing. -Produce 16Mb/ run/ 6h -Scheme: DNA sequence > fragments > turned into ssDNA > go to the chip > one sequence per well > amplification occurs with wells being embedded by one base at a time > base incorporation emitted light > laser read and compute 14)About Solid sequencing: -Sequential ligation sequencing -New: instead of using an elongation process, it is based on ligation process. A dinucleotide is ligated once at time and each dinucleotide or dibase probe presents a different radiolabeled tag. Ligation increase light and then accuracy. Total of 4 dyes, each dye represents 4 of 16 possible dinucleotide sequences. -Two ways of sample preparation: (1) single DNA molecule which has 2 adaptors inserted, one at each end (2) two DNA molecules, called mate paired, that have a total of 3 adaptors, one in each end and one in the middle. -Scheme: DNA sequence > fragments > adaptors > beads > amplification > beads in slide glass > ligation of dibase probe > each incorporate dibase release specific light > captured by machine -Number of samples: 1, 4 ,8 samples /glass or with barcoding it can be up to 250 samples. -Advantages: (1) increased accuracy (2) superior detection of SNP 15)About Ion torrent sequencing: -New: instead of using radiolabeled bases, it uses changes in pH during incorporation of a base. -Advantage: use less memory to process information -Faster technique: full run 2-4h, reads 200-400bp, produce total 10Gb -Scheme: DNA sequence > fragments > adaptors > selection > beads > a+plification > bead in each well > well is fulfilled one base pair at a time > incorporation release H > change pH > changing in pH results in changing voltage > machine capture voltage > record the base that produced the changing in pH > if more than one of same base is incorporated, more H is released so machine is able to tell how manybases were incorporated in a repeat mode 16)About Nanopore sequencing: - Theory: tiny hole in a membrane can measure tell what base is passing though by its structural characteristic.So you can useaβ-hemolisinmembraneorMicrosporidium MnpAporeovera biolayer membrane as a pore in which your DNA molecule will pass. - The pore can be synthetic or not but at maximum a diameter of 1.5 nm. - Oxford Nanopore is the main group working on that. - Also called Strand sequencing - Scheme: membrane with a protein pore and a potential protein inside the pore that measures the molecule that pass though the pore and also slow the flow, over the pore, a DNA enzyme complex responsible to hold that DNA molecule and guide it through the pore and also unzip the DNA > one side of the chip is inserted the buffer containing the DNA molecules > other side of the chip contain plain buffer > voltage is applied to the system so it guides DNA to go through the pore > DNA enzyme complex separate the strands but also hold the strands so when one strand passes, the other one start going - Variation of the method included (1) endonuclease enzyme, which would be cutting each base at time and leaving it to pass through the pore. It did not work as much because it slows down too much the process and sometimes the base could be lost instead of passing through the pore. (2) minion which is a chip that has a USB port so the reads can be transferred and analyzed in a laptop after the processing. - Most important characteristics: (1) do not need primers, polymerase, amplification (2) do not need chemical process or chemical denaturation (3) can get long sequences as 200Kb (4) no fluorescent or radiolabeled tags (5) no laser or pH - Possibly the best future in sequencing that would be available for all. PART 4: PROKARYOTES 1) Themain characteristics ofProkaryote: (1)single cell unit (0.2-10µm),reallysmall (2)nomembrane bond organelle, which means they don’t have mitochondria, chloroplast, endoplasmic reticulum, Golgi, etc (3) simple structure, however complex chemical structure that allows them to survive in extreme environments. 2) Main domains include (1) Archaea, which is closer related to Eukaryote than Bacteria (2) Bacteria, which are more specialized than Archaea. Earth originated around 4.5 bya. Prokaryote originated (by fossil proves) around 3.5 bya (billion), which means that they appear pretty soon on Earth and got adapted to extreme environmental conditions as lack of oxygen and presence of toxic substances and high temperatures. 3) Ways of living (1) autotrophs: produce energy by photosynthesis (2) heterotrophs: live in/on another organism – host- gain substance from other creatures. Heterotrophs can be classified as: (a) commensal, live in /on another organism without causing harm or benefits (b) symbionts or mutualism, live in/on another organism in a mutual beneficial relationship (c) parasites, live in/on another organism being detrimental to the host. Symbionts include (1) nitrogen fixing bacteria living at roots of some plants (2) methane producing bacteria from herbivore intestinal tract (3) microbiota of many animals. 4) Oldest bacteria are anoxygenic photosynthesis. They don’t use oxygen/ water to get ion for metabolism. They use H S2 5) Characterization of Prokaryote: (1) before it was based on physiological and morphological findings (2) nowadays it is based on differences at 16S – ribosomal RNA gene (3) with increasing in molecular technologies probably on future it will be based on whole genome sequencing or even entire sequencing of 16S. 6) Up to now, ~9,300 prokaryote organisms were described but it is estimate that up to 4 million exists. Bacteria is the domain that is more described, almost 10 times more species described than Archaea. 7) Characteristics of Prokaryote genome include: (1) circular (2) single large chromosome molecule (3) extra chromosomal elements at cytoplasm, such as plasmids (4) collinear structure (5) no introns. 8) Extra chromosomal elements are DNA molecules that stay out of the main chromosome region. Usually they have its own mechanism of replication which is independent on cell replication and division. They are considered part of whole genome once some of them contain housekeeping genes important for the organism survival. Eukaryotes can have extra chromosomal elements. 9) Genome size is the sum of length/mass of a single copy of a cellular DNA molecule regardless its function. It can be difficult to estimate once some prokaryote keep more than one copyof its genome even without being on division. 10)About bacterial structure: Pili is a fimbria which can be used for (a) adhesion or (b) conjugation, in case of specialized F pilus. Plasmid is an extra chromosomal element. Capsule is a carbohydrate coat that stays over the cell wall. Also called slime. All bacteria have cell wall, except Mycoplasma. Cell wall is formed by peptidoglycan membrane that allows bacteria to be classified as (1) Gram +: have thicker peptidoglycan that adsorbs crystal violet and get blue stain (2) Gram -: thinner cellwallso crystalviolet isnotadsorbedbutthese bacteriahaveanothercoveringlayerthatallowsstaining with lucipherin and these one includes the most resistant and pathogenic bacteria. Nucleiod is the region where the chromosome of the prokaryote is localized. It is formed by 80% DNA and 20% protein. Ribosomes from prokaryote are smaller than eukaryote. Bacteria have the 70S ribosome. Flagella allows movement of cell and can be classified as (1) polar or (2) peritrichious. Few Bacteria are pathogenic, however the ones that are, they are the most described and studies ones. About bacteria genome: -DNA is packed by proteins and chemical molecules. Even destroying the genes responsible for packing the DNA, it keeps structurally hold together and seems to not interfere on survivorship, which means that it has unknown molecules involved on its packed and expression. -Chromosome is organized by loops of ~40kb in length, each loop is anchored by a type of protein (RNA?) complex. -Nucleoid integrity is kept by protein, loops and other chemical molecules. Experiment doing osmotic lysis of cellular membrane showed that DNA did not got totally spread which is another prove that unknown elements are responsible for packing and keeping DNA in a close region called nucleoid. AnotherexperimentdidnicksonDNAmoleculeanditdidnotinterferewithpacking,DNAdidnotspread on cytoplasm, which means again, unknown structures hold them together. -Genome of bacteria is fold ~1,000 fold. -Nick is a break of only one strand of the DNA molecule while break is when both strand are disrupted. 11)About Archaea: - Uniqueness: they have a chromosome structure and organization so different that it comes to the question if they should be considered a separate domain. - More close relate to Eukaryote than Bacteria. From 27 characteristics, they share 8 with Eukaryote, while Bacteria share only 3. - Membrane ether-linked, which is different from Bacteria that is ester-linked membrane. - Don’t have murrain on the cell wall, no peptidoglycan. - They can live in more extreme environments than bacteria which include organisms that are (a) thermophilic – high temperature (b) acidophilic – high acidic Ph (c) halophilic – high salt concentrations - Many are dependent of sulfur and poisoned with oxygen - Their chromosome is organized in nucleosome just like Eukaryote. They have histones, positively charged proteins that pack DNA. There are two main histones (a) HmfA which is more abundant in log phase (b) HmfB that can form byitself a tetrameric structure to hold the DNA, prettyclose to (H3- H4) 2rom eukaryote. HfmA and HmfB are present in equal amount during stationary phase. - Nucleosome is responsible for pack 80bp of DNA. - Archaeal nucleosome may contain different histones depending on growth condition. 12)Chromosome is a cytochemical definition in which is considered chromosome molecules that can stain with specific markers. However, because of prokaryote chromosome organization it does not stain. Chromosome is found at the nucleoid region that is made up of DNA molecule and proteins. 13)Disruption by osmotic experiment shows that DNA comes out of the prokaryote in coils and interacting with proteins. 14)Organisms with smallest genome are usually heterotrophs, specialized organisms that live in/on host, just like parasites while organisms with largest genome are usually generalists and autotrophs. 15)Large genomes are often composed by (a) repetitive regions (b) duplications. 16)Exceptions ofprokaryote genome(a)linearchromosome(b)duplicatechromosome – morethanonecopy, not true haploid (c) two or more DNA molecule on chromosome 17)Supercoiling is the effectively coiling of a coil (helix). It is used to control DNA expression and fold it to fit inside the cell. Topoisomerase is the enzyme responsible to reduce supercoiling of the DNA, so it can be transcribed or replicated. In a gel of agarose, relaxed, linear DNA can be found close to anode. As the DNA is cut bytopoisomerase to reduce the supercoil, more bands representing relaxed strands are formed. Less supercoiled DNA are shownat thirdgel in which small bandclosetothecathodeis shown.SupercoiledDNArunsfasterthrough the gel because it is small and goes through the pores faster. 18)About Lac Operon: -operon is a region of the chromosome that produces a mRNA which includes more than one gene together. Eukaryotes do not have operons. -Lac operon is responsible for the metabolism of lactose and includes in total 3 genes: (a) LacZ that translate the βgalactosidase responsible for turning lactose into galactose and glucose (b) LacY that translate the lactose permease responsible for bind to cellular membrane and put more lactose inside the cell – increase permeability of lactose (c) LacA that translate transacetylase, not involved on lactose metabolism. -other elements for this region includes (a) LacL which produces a tetrameric protein also called Lac repressor responsible for blocking the RNA polymerase to transcribe the Lac genes – operon – when lactose is in low concentration (b) promoter region down the LacL where RNA polymerase recognizes (c) operator, region in which Lac repressor binds to block RNA polymerase. -at high concentrations of lactose, lactose serves as inhibitor, also called inducer, of LacL tetrameric protein. It binds and change conformation of Lac repressor that no more can bind to operator region so now RNA polymerase can transcribe the operon. As lactose is metabolized, it decreases it concentration allowing Lac repressor to binds again to operator. -discovered/described by Monod and Jacob. -importance of this process: first described way of gene expression and controlling. It was the first model for gene regulation. It is a primeexampleofnegativefeedback. AlthoughEukaryotedoes not haveoperon, it is known that other molecules also acts as inducers and repressors, such as transcriptional factors that often acts as positive regulation of gene. They bind to regions of genome that is going to be transcribed, some are responsible for recruiting RNA polymerase to the gene or increase transcription (enhancer regions). 19)About plasmids: -definition: extra chromosomal elements found in the cytoplasm of the bacteria. They are independent DNA molecule which can replicate byitself and have its own regulation (have their own replication start sites). Usually circular. -Only few bacteria have plasmids. -Classification of plasmids can be based on: Function Transfer Properties Types of DNA transfer (1) R – antibiotic resistance (1) Self-transmissible: (1) Donation: when a gene: it was the first plasmids that are conjugative plasmid can described gene due to the conjugative and provide conjugative pharmacological mobilizable. Usually F function for a mobilizable importance. Bacteria plasmids. plasmid so both can be without positive selection (2) Non-transmissible: transferred. pressure can lose this plasmids that cannot initiate (2) Conduction: when a self- plasmid! contact with recipient transmissible plasmid (2) F – fertility factor: largest bacterium neither prepare recombines with non- type of plasmid and allows the DNA for transfer. transmissible plasmid it is transfer. ~9.5 kb in (3) Conjugative: able to initiate transferring a co-integrate E.coli. contact with recipient molecule. (3) Col – colicinogenic gene: bacterium. (3) Transduction: when a responsible for translating (4) Mobilizable: able to phage is used as a vector to the colicin protein that prepare the DNA from transfer a bacterium genetic blocks the growth of plasmid for transfer. material from a donor surrounding bacteria Sometimes a bacterium needs bacterium to a recipient. Plasmids can be R and Col in more than one plasmid to be the same time. able to transfer it. Usually Coil and R plasmids are either simply mobilizable, conjugative and mobilizable or non-transmissible. -because plasmids are self-replicated and have own ways of gene expression, but same structure as bacterial genome, it was hypothesized that they were parts of bacterial genome that got rid of the main chromosome. -plasmids can be transferred from one to another bacterium by horizontal transfer. Horizontal transfer is when genetic material information is passed through individuals without hereditary relationship, without genera
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