nucleic acid synthesis
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89 terms
English | Photos |
|---|---|
 Nucleic Acid primary structure | -nucleotide sequence |
| Difficulty determining nucleic acid primary structure | -contain only 4 unique monomeric units -with only 4, there are fewer specific sites for cleavage -distinctive sequence more difficult to recognize -greater likelihood of ambiguity -many monomeric units in most polynucleotides |
| Whats the two breakthrough that made sequencing of nucleic acid easy? | -discovery of restriction endonuclease -polyacrylamine gel electrophoresis |
| polyacrylamine gel electrophoresis | -separation methods to resolve nucleic acid fragment that differ from one another in length by just one nucleotide |
| Whats the use of restriction endonuclease in sequencing nucleic acid? | cleave DNA at specific oligonucleotide sites, generating unique fragments of manageable size |
| What 2 basic protocols for nucleic acid sequencing are in widespread use? | -Chain termination or dideoxy method (Sanger) -base-specific chemical cleavage method (Maxam and Gilbert) |
| How are Sanger and Maxam-Gilbert method similar? | -carried out on nanogram amount of DNA -DNA chain is detected -electrophoretic separation on polyacrylamine gels -typically labeled with radioactive P32 -pattern of separation visualized by autoradiography |
| Sanger method | -dideoxy sequencing or chain termination -based on the use of dideoxynucleotides (ddNTP's) in addition to the normal nucleotides (NTP's) found in DNA 1- dsDNA are denature by heat turning into ssDNA 2-primer annealed at one of template strand (primed synthesis method) 3-primer or one of nucleotides are radioactive or fluorescent so final material can be detected on gel 4- the solution is divided into four tubes labeled "G", "A", "T" and "C" 5-all of the tubes contain a different ddNTP present, and each at about one-hundreth the concentration of the the normal precursors 6-DNA is synthesized, nucleotides are added by the DNA polymerase; on occasion a ddNTP is incorporated into the chain in place of a normal nucleotide, which results in a chain-terminating event 7- newly synthesized dsDNA is once again denature and in preparation for electrophoresis 8-each tube runs in a polyacrylamine gel and are separated by band sizes 9-DNA now may be sequenced from small to big strands |
| Dideoxynucleotides | -essentially the same as nucleotides except they contain a hydrogen group on the 3' carbon instead of a hydroxyl group (OH) -These modified nucleotides, when integrated into a sequence, prevent the addition of further nucleotides |
| Why does dideoxynucleotides terminated DNA chain ? | -because a phosphodiester bond cannot form between the dideoxynucleotide (lacking 3'-OH) and the next incoming nucleotide(5'), and thus the DNA chain is terminated |
| Sanger's tubes | "G" tube: all four dNTP's, ddGTP and DNA polymerase "A" tube: all four dNTP's, ddATP and DNA polymerase "T" tube: all four dNTP's, ddTTP and DNA polymerase "C" tube: all four dNTP's, ddCTP and DNA polymerase |
| Example of Sanger Sequence for "G" (img) |  |
| (T/F)Dideoxy sequencing use the same DNA strand for all the reaction with different ddNTP. Why? | -True -because new chain will terminate at all positions where the nucleotide has the potential to be added because of the integration of the ddNTP. Making full sequencing possible |
| How does Sanger methods keeps termination random? | -incorporate ddNTP infrequently -four reactions are run, one for each ddNTP (termination can be random and occur everywhere in the sequence) |
| Reading dideoxy sequencing gel (img.) |  -visualized by autoradiography, followed by separation according to size by polyacrylamide gel electrophoresis -read from bottom to top, since smallest fragment travel faster (5>3) -convert "sequence" into complementary sequence and find unknown original sequence |
| Main difference between Sanger and Maxam-Gilbert method | Sanger extends, while Maxam-Gilbert cleaves |
| Whats the most used sequencing method? | -Sangers |
| Automated DNA sequencing | -campable of identifying 10⁴ bases per day -use fluorescent dye of different colors to label primer DNA for all 4 sequencing reaction -they can all be run together -with low power laser bean can detect primer hence base immediately -u would still take about 8 yrs to sequence a whole genome with 100 machines at fool speed |
| DNA replication | -the sequence of nucleotides in one strand is copied in a complementary fashion to form a new second strand by the enzyme DNA polymerase -each strand template for copying -DNA polymerase requires template or primer -DNA polymerase add nucleotide in a 5' > 3' fashion |
| Primer | A oligonucleotide with a free 3' end, bound by complementary base pairing to the template strand, that is elongated during DNA replication by DNA polymerase |
| DNA secondary structure | -A B Z -all sugar-phosphate backbone outside -base (H bond) inside -minor and major groove - π-electron cloud, hydrophobic on flat side |
| Canonical base pair | -A:T and G:C have nearly identical dimension -Sugar-phosphate backbone also have H bonds |
| B-DNA | -ladderlike hypothetical conf. are unfavorable because water could have access to bases -ladderlike convert to helix by w little right hand twist -twist bring stack close together 0.34nm apart, w/out affecting sugar-phosphate 0.6nm distance -longer, thinner, right handed -10bp, 3.32 A (rise per pair) -anti glycosidic bond conf. |
| A-DNA | -right handed -2.3 A rise per pair -11bp -shorter, broader -bases displaced around -dehydrated DNA fiber -anti glycosidic bond conf. |
| Z-DNA | -longer, slimmer -3.8 A rise per turn/ left handed -12 bp -syn G and anti C, but whole C nucleoside (base +sugar) flips 180° -found in C:G rich region of DNA |
| propeller twist | -allows greater overlap between successive bases along a strand of DNA and diminishes the area of contact between bases and solvent water. |
| (T/F) It is topologically possible for the G to go syn and the C nucleoside to undergo rotation by 180° without breaking and re-forming the G:C hydrogen bonds on Z-DNA | True |
| Details about C:G H-bond in Z-DNA | -N-glycosyl bonds of G residues in this alternating copolymer are rotated 180° with respect to their conformation in B-DNA, so that now the purine ring is in the syn rather than the anti conformation -pyrimidine flips their nucleoside 180° -G:C H-bond not broken -as a consequence of these conformational changes, the base pairs in the Z-DNA region no longer share p , p stacking interactions with adjacent B-DNA regions. |
| Why pyrimidine does not adopt syn conformation in Z-DNA | -pyrimidine nucleosides do not readily adopt the syn conformation because it creates steric interference between the pyrimidine C-2 oxy substituent and atoms of the pentose. -Because the cytosine ring does not rotate relative to the pentose, the whole C nucleoside (base and sugar) must flip 180° |
| how many base-pair segment of B-DNA is converted to Z-DNA through rotation of the base pairs? | 6-base-pair segment of C:G |
| What do they mean by Z-DNA dinucleotide? | Because alternate nucleotides assume different conformations, the repeating unit on a given strand in the Z-helix is the dinucleotide. -for any number of bases, n, along one strand, n-1 dinucleotides must be considered -GpCpGpC subset of sequence along one strand is comprised of three successive dinucleotide units: GpC, CpG, and GpC |
| The conformational alterations going from B to Z realign the sugar-phosphate backbone along a _______ course that has a ____-handed orientation thus the designation Z-DNA. | -zigzag -left handed |
| Note that in any GpCpGp subset, the sugar-phosphates of GpC form the ______"zig" while the CpG backbone segment forms the _______ "zag." | -horizontal -vertical |
| (T/F) Z-form can arise in sequences that are not strictly alternating Py-Pu | True |
| Denaturation of DNA | -pH, temp, ionic strength all disrupt H- bond -double helix denature -helix melt about 80°+ - |
| Does UV absorbency increases or decreases as base unstack? | increases by 40% |
| Stability of double helix | -internal and external H-bond -the external are the phosphate and water molecule - P⁻ are situated in a way that they have minimal effect one another and are free to interact with cations such Mg²⁺ |
| hyperchromic shift | -absorption increase due to denaturation - Unstacking alleviates this suppression of UV absorbency -rise in absorbance coincides with strand separation |
| Melting temperature Tm | -midpoint of absorbance increase on hyperchromic shift due to strand separation |
| Why does DNAs differ in their Tm values? | because they differ in C+G content -the higher the [ ] the higher the melting temperature (due to H-bond) |
| The _____ the ionic strength, the _____ the melting temperature. why? | -lower -lower -Ions suppress the electrostatic repulsion between the negatively charged phosphate groups in the complementary strands of the helix, thereby stabilizing it. |
| (T/F)DNA in pure water melts even at room temperature. | True |
| What happens to DNA double helix if concentration of ion is raised? | -Tm raises -transition between helix and coil is sharp |
| Renaturation | -The process by which 2 complementary single-stranded DNA molecules pair; also called reannealing -the rate of renaturation is an index of DNA complexity -Many of the realignments are imperfect, and thus the strands must dissociate again to allow for proper pairings to be formed -process is faster if temp is warm to promote diffusion |
| If DNA is real complex, the reannealing process is fast or slow? | slow |
| Thermal denaturation and renaturation of DNA | .-nucleation phase of the rxn is a second-order process depending on sequence alignment of the two strands. -slow process cuz it timely for complementary sequence to encounter one another in solution then align themselves. -Once the sequences are aligned, the strands zipper up quickly. |
| Supercoil | -one type of tertiary DNA structure with high order folding -overwound or underwound -allows easy manipulation and compact storage -relies on topoisomerase |
| When and where is supercoil found? | -Double-stranded circular DNA (or linear DNA duplexes whose ends are not free to rotate), form supercoils if the strands are underwound(negatively supercoiled) or overwound (positively supercoiled) |
| Negatively supercoiled | -DNA is underwound -underwound duplex DNA has fewer than the natural number of turns -once DNA POL III has duplicated the strand, its somewhat loose, and the release of tension bu underwound favors writhes B-DNA supercoil into the right hand direction. |
| positively supercoiled | -overwound DNA has more than then natural number of turns -happens before DNA polymerase passes through it in the replication process -the formation of bubble by helicase add twist to double stand ahead, and in order to release tension, the dsDNA supercoil into writhes in the left hand direction just like DNA helix |
| Whats the lowest energy state in B-DNA ? | 10bp per turn of its helix |
| Why supercoiling does not work on free end DNA? | because if top supercoil, bottom uncoils giving it balance by distribution of stress |
| Bacteria or eukaryotes DNA usually folds into loops stabilized by proteins and supercoil takes place withing the loop. | both -in Eukaryote protein prevents free rotation of the ends) |
| torsional stress | -rotation about single bond causing steric hindrance -happens in supercoiling of duplex DNA |
| topoisomerase | -torsional strain makes DNA unstable and in order to lower energy require to maintaining structure, # of twist and writhes are minimized -enzyme that can add and remove rotation from DNA helix temporarily breaking nucleotide strand, swirling or untwisting the end, and rejoining the broken ends -type I or II -Gyrase and helicase = Type II |
| Gyrase | -type II topoisomerase -introduces negative supercoil -this enzyme break both H bonds and sugar-phosphate backbone pass strand to to other end, and rejoin |
| Helicase | -type II topoisomerase -uses ATP -does not alter linking number -negative coiling behind it -an enzyme that opens bubbles in small segments of the DNA double helix during replication by breaking hydrogen bonds building tension and inducing positive supercoiling which is release by gyrase |
| Type I topoisomerase | -Relaxes supercoils by forming single stranded breaks (Swivelase) then rejoining them |
| Type II topoisomerase | two DNA strands are cleaved, duplex DNA is passed through the break, and the break is resealed. Type II topoisomerases therefore change the linking number in increments of two rather than one |
| Twist | -number of helical turns |
| Writhe | -the number of supercoil -can be negative or positive -most cells are negative supercoiled, meaning underwound, with writhe in right hand direction |
| Linking number | -L = T + W -number of times the two strands are intertwined, and, provided both strands remain covalently intact, L cannot change. -values of T and W for various positively and negatively supercoiled |
| 2 advantages of supercoiling in DNA | -make separation of two strand easier and faster during replication and transcription, since its requires less energy -can be packed in smaller places |
| Bacterial Gyrase | -bacterial enzyme that induces negative supercoiling -T tend to decrease due to negative supercoil caused by torsional stress -usually W is reduced and T decreases slightly, and changes are distributed evenly. |
| Cruciform | -inverted repeat DNA sequences called palindromes -when normal interstrand base pairing is replaced by intrastrand -form hairpin -not as stable as DNA ds due to unpaired end loops -two-fold rotation symmetry about their center |
| Palindromes | -sequences of DNA that are identical when read from 5' to 3' direction on one strand and the 3' to 5' on the other strand -may form cruciform |
| Cruciform (img) |  |
| Chromosome Structure | -human DNA ~ 2 meters -packing made possible by wrapping of DNA around nucleosomes and helical filament conformation -helical filaments arranged in loops associated with nuclear matrix |
| Nuclear matrix of chromosome structure | -skeleton or scaffold of protein, providing structural framework within nucleus |
| Nucleosome | -, The basic, bead-like unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound around a protein core composed of two copies of each of four types of histone. |
| Histone | -A small protein with a high proportion of positively charged amino acids that binds to the negatively charged DNA and plays a key role in its chromatin structure. -arginine and lysine -H1, H2A, H2B, H3, H4 |
| Histone octamer structure | -made of 2 pairs of H2A and H2B, H3, H4 to form nucleosome core -rich in (+)charged arginine and lysine which interact with P⁻ of backbone |
| Nonhistone proteins | -found in chromatin -very few compared to histone -regulator of gene expression |
| Chromatin |  Granular material visible within the nucleus; consists of DNA tightly coiled around histone forming nucleosome, which in turn are wound in solenoid fashion, having 6 nucleosome per turn, forming DNA loops. . |
| Nuclear matrix miniband |  -nucleosome solenoid loop bases are attached to nuclear matrix that arrange itself in a circular form as a miniband unit of chromosomes -stack in each chromatid of human chromosome |
| Sec/ Tert Structure of RNA | -RNA often forms intrastrand H-bonds complementary sequence -cannot form B-DNA type double helix due to 2' -OH group -conf similar to A-DNA -11 bp per turn, base strongly tilted from the plane perpendicular to helix axis -ex: tRNA and rRNA |
| tRNA secondary structure | -each cloverleaf has 4 H-bonded segments , 3 loops and stem where 3' and 5' end -only one tRNA structure is known -a lot of invariant residues, which for the most part lie in tRNA non H-bonded region |
| What are the 4 tRNA segments | -acceptor stem -D loop -anticodon loop -TΨC loop |
| tRNA acceptor stem role | -a.a. donating species in protein synthesis -thats the stem 3' and 5' -the 3' end -CCA combine with -COO⁻ from a.a. |
| D loop | -one of the 4 segments of secondary tRNA structure -named so due to dihydrouridine base |
| Anticodon loop |  -The portion of a tRNA molecule that contains the anticodon seq. which base-pair with the matching codon sequence in mRNA during translation. -there are 7 unpaired bases on this loop -reading 3' > 5' anticodon is preceded by an alkylated purine and followed by U base |
| Variable loop | -also called extra -varies from tRNA to tRNA -from 3'> 5' btw the TΨC loop and anticodon loop |
| TΨC loop | -several unpaired bases including (Ψ)- pseudouridine -ribosome binds to tRNA by Ψ recognition |
| tRNA 3' end (img) |  |
| tRNA tertiary structure | -H bonding btw bases on the D loop, variable and Ψ loop -H-bonging involves invariant nucleotids of tRNA, showing its important in 3-D structure |
| Which tertiary structure in RNA involves many H-bond with base pairing that are non -canonical? | -tRNA tertiary structure |
| What gives the stable L shape tert form to tRNA? | the H-bonds between D and Ψ arms folding them together |
| L shape tertiary tRNA structure (img) |  |
| rRNA |  -large degree of intrastrand sequence complementary -highly folding pattern allow such complement to happen with each other -structure is rich in short, helical segments separated and punctuated by single stranded loops |
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