Flashcards in DNA Chemisty And Analysis Deck (64):
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DNA and RNA
-composed of nucleotides linked in linear chains (polymers of nucleotides)
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Nucleotide
-Nitrogenous base- purine or pyrimidine
-Sugar moiety-ribose or deoxyribose
-Phosphate
Nucleotide-phosphate in group
Nucleoside- base of sugar minus phosphate
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Rare or minor bases
-methylation, acetylation, and hydroxymethylation
Transfer RNA contains high percentage of minor bases
Ribosomal RNA contains high percentages of methylated bases
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Methylation
-methyl group acts as silencer of gene expression
--5-methylcytosine is the most common modified base in DNA, most often CpG sites are modified
-cytosine loses amino group and becomes Uricil (deamination). Would be damaging to DNA, this is why DNA has Thyamine
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DNA contains:
A. Nitrogenous bases: adenine, guanin, cytosine, and thymine.
B. Sugar moiety: 2-deoxyribose
C. Phosphate
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RNA contains:
A. Nitrogenous bases: adenine, guanine, cytosine, and uracil
B. Sugar moiety-ribose (hydroxyl group at 2nd position)
C. Phosphate
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Linkage of Nucleic acids
-mononucleotides are joined by phosphodiester bridges between the 5'-hydroxyl group of one nucleotide and the 3'-hydroxyl group of the adjacent nucleotide.
-bases are not directly attached covalently to one another
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Nuclei acid sequences read:
-left to right in the 5' to 3' direction
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Acid hydrolysis
-products of strong acid hydrolysis of DNA or RNA are purine bases, pyrimidine nucleosides, deoxyribose or ribose and phosphate.
-Purine nucleoside are acid labeled while pyramiding nucleosides are stable in acid (will lose purines)
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Base hydrolysis
-RNA proceeds through a 2', 3'-cyclic phosphate intermediate.
-DNA doesn't contain a 2'-hydroxyl group, it is resistant to base hydrolysis (important method for separating DNA from RNA)
--DNA is stable in base and RNA is not stable.
-product of base hydrolysis of RNA are 2' and 3' nucleoside monophosphates.
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DNA Double Helix
-prokaryote and eukaryotic DNA exists as a double helix
-right handed helix (alpha-helix)
-phosphodiester bonds and the deoxyribose moieties form the backbone of each strand and nitrogenous bases project into the interior
-nitrogenous bases:planes of bases are parallel to each other and perpendicular to the long axis of the double helix. van der Waals forces greatly stabilize the helix
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Polar groups
-phosphates project to the outside where they interact with proteins (ones carrying + charge at physiological pH)
-negatively charged phosphates (at physiological pH) are complexed with cations such as Na, K, and CA in vivo-leads to stabilization
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Nonpolar groups
-interior of the molecule is very hydrophobic-water is excluded
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Base pairs
-hydrogen bonds between base pairs hold strands together
A-T and G-C
-number of A=T
-number of G=C
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Frequency of base pairs
A=T does not necessarily equal G=-C
-GC has three hydrogen bonds compared to AT which has two, so GC is stronger
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Antiparallel
-one strand runs in the 5' to 3' direction while the other strand runs in the 3' to 5' direction
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X-ray crystallography of DNA and distances
Distance b/t stacked nitrogenous bases (0.34 nm) and the distance for one complete turn of the double helix (3.4 nm)
-10 base pairs for each complete turn of the double helix.
-B form is most abundant
-10A= 1nm
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Grooves
-grooves in the DNA (particularly the major groove) provide surfaces to which regulatory proteins can bind.
-major grooves regulate genes (turn on and off)
-major groove is almost twice as wide as the minor groove (22A versus 12A)
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Reasons for DNA stability
-hydrophobic and electronic interaction b/t the stacked bases of each strand.
-hydrogen bonds b/t strands
-hydrophobic nitrogenous bases are shielded from the aqueous environment
-negatively charged phosphate groups and polar sugar moieties are exposed to the aqueous environment.
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Melting
-heating DNA disrupts hydrogen bonds and the strands separate.
-AT melt at lower temp than GC
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Hypochromic effect
-melting of DNA increases ultraviolet light absorbance
-stacked bases in double stranded DNA decreases the UV absorption relative to free nucleotides in solution.
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Annealing
-opposite of melting
-hydrogen bond formation allows double strand DNA to form two complementary single strands
-slow step
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Conformations of double helical DNA
--changes in the shape of DNA may function in regulation of gene expression, since conformational changes in DNA can affect the binding of proteins to DNA, and protein binding is essential to regulation.
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B-DNA
-most common conformation
-discovered by Watson and Crick
-right turn
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DNA C
-9 base pairs per complete turn of the double helix with the distance for one complete turn being 3.3 nm.
-not thought to occur in vivo
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DNA A
-11 base pairs per complete turn of double helix with distance being 2.8nm
-nitrogenous bases are not perpendicular to the long axis of the double helix but have a tilt of about 20*
-forms under low hydration conditions
-occurs in very short stretches in DNA and is DNA sequence dependent
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DNA Z
-has 12 pairs per each complete turn of the double helix
-observed in crystals of synthetic hexanucleotide d (CpG)3 and fibers of the alternating d(GC)n polymers
-has left-handed rather than a right-handed helix axis of rotation of the DNA coil opposite in the B (and A)
-backbone has a staggered zigzag course
-may exist in DNA in vivo, in stretches of alternating guanosine-cytosine or in other kinds of alternating purine-pyrimidine sequences
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DNA within cells
-most is in B conformation
-regions rich in GC base pairs may assume the Z conformation
-methylation of deoxycytidine groups in the G-C rich regions of DNA may favor Z-DNA formation
-possible for a B-Z transition to occur within segment of DNA without totally separating the two strands
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Short stretches of adenine
-can cause bending of the DNA helix
-6 adenines in a row can cause a bend of about 18deg. Bending may be a feature recognized by DNA binding proteins. The expression of genes is generally blocked in regions of DNA with an A-bending sequence
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Coding strand
-nucleotide sequence in the coding strand is the same as that of the RNA made from the DNA, except uracil will substitute for thymine in the RNA
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Template (noncoding) strand
-strand is complementary to the coding strand and also to the RNA which will be synthesized.
-if the two strands of DNA were separated, the newly synthesized RNA would bind (hybridize) to the template strand but not to the coding strand
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Denaturation (melting)
Factors?
-separation of the two strands of DNA molecule due to disruption of the bonding forces between the strands.
-high temperature
-high pH
-low ionic strength->negative charges on the two strands will repel each other
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Significance of Denaturation
-necessary component of both DNA replication and RNA synthesis.
-desaturation requires energy expenditure to occur in vivo
-helpful in developing experimental approaches for diagnosis of various diseases
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Renaturation (Hybridization)
-separated DNA can reform a double helix if appropriate conditions are provided
-Filter hybridization
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Southern blot (E.M. Southern)
-DNA sample is electrophoresed
-denatured (the strands separated)
-transferred to nitrocellulose and hybridized with a radiolabeled probe (radiolabeled DNA or RNA)
-technique is often used to see gene structure or gene complexity
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Northern Blot
-RNA sample is electorphoresed, transferred to a membrane and immobilized, and then hybridized with a DNA probe
-technique is used to measure RNA message levels for genes
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Western Blot
-protein sample is electrophoresed, transferred to a membrane and then visualized by an immunological procedure (antibodies)
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Nucleases
-hydrolyze DNA and RNA
-RNases hydrolyze RNA
-DNases hydrolyze DNA
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Exonucleases
-enzymes that degrade DNA or RNA one base at a time starting from either the 5' or 3' end of the molecule
-EDTA will chelate exonuclease so DNA you're working with wont be eaten up
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5' exonucleases
-must be presented with a 5'-hydroxyl on the terminal nucleotide to remove it from the polynucleotide chain
Ex: spleen phosphodiesterase
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3' exonucleases
-must be presented with a free 3' OH on the terminal nucleotide to remove it from the chain
ex: nuclear activity of DNA polymerase I, snake venom phosphodiesterase
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Endonucleases
-enzymes which hydrolysis in the interior of a polynucleotide
-some are specific for the 5' side of the phosphodiester bond and some for the 3' side.
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DNase I
-endonuclease which cuts double-stranded DNA in a nonspecific fashion
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Restriction enzymes
-endonucleases that recognize specific base sequences in double-helical DNA and cleave both strands of the duplex DNA
-found in a wide variety of bacteria and they are called restriction enzymes because they restrict the growth of bacterial viruses (bacteriophages)
-valuable tools for analyzing chromosome structure, creating recombinant DNA molecules, isolating genes and sequencing very long DNA molecules
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Palindrome
-phrase (sequence) which reads the same ways forwards and backwards.
-recognition sequences for most restriction enzymes are palindromic
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Vector-not really a definition
-DNA fragments from the organism of interest (human, mouse, sea urchins) are linked to a vector, a bacterial, or viral fragment of DNA which can be propagated in the appropriate host organism
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Cloning human gene into bacterium
-fragment of DNA obtained from a genomic or cDNA library is introduced into a plasmid (small, circular DNA present in many bacteria).
-plasmid is reintroduced into a bacteria (called transformation)
-bacteria possessing the "engineered plasmid" are identified (called selection) and isolated.
-bacteria containing a specific cloned fragment of DNA are referred to as a "clone"
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Desirable Vector Characteristic
-posses a number of sites which a re cleaved by different restriction enzymes (multiple enzyme choices)
-bears genes which code for proteins that impart resistance to antibiotics. Allows for selection of bacteria into which the construct has been inserted
-possess internal signals (promoter squelches) which permit the insert to be transcribed and subsequently translated into the eukaryotic protein of interest
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Sticky ends
Ligation
-through hydrogen bonding complementary extensions from enzyme digestion facilitate joining of DNA fragments by ligation.
-ligation is an enzymatic joint of DNA by phosphodiester bonds
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Transformation
Plasmid is reintroduced into a bacteria
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Vectors
-size of the piece of foreign DNA that can be inserted into plasmids is small (6-10 kb)
-lambda phage vectors->can accumulate 20 kb
-COSmids are plasmids which contain DNA sequences (called cos sites) that allow phage DNA to be packaged into a bacteriophage protein coat. Allows efficient expression of the library. DNA insert can be up to 50 kb in size for COSmids
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Genomic DNA Libraries
-constructed by cloning all the DNA from the genome of an organism.
-usually use lambda phage vectors that tolerate large inserts so fewer clones are required to obtain entire genome
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Highly repetitive DNA sequences
-localized in the centromere region of the chromosomes
-do not appear to be transcribed
-referred to as satellite DNA
-Alu I family of repetitive DNA occurs hundreds of thousands of times in the human genome. May have a role in recombination of genes. Only found in primates
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Moderately repetitive DNA
-consists of both transcribed and untranscribed sequences (97%+ of human DNA has no known function, yet)
-ex: ribosomal RNAs, transfer RNAs, and five classes of histone proteins.
-number of copies of these genes per genomes can range from several hundred to several thousand.
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DNA size
-haploid cells contain approximately 3.5 x 10^9 base pairs of DNA
~25,000 genes are estimated to exist for humans.
-up to about 10,000 different proteins might be made as a given time in any one cell, a very small percentage of the genome (~1%)
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Chromosomes
-DNA is located
-each chromosome contains one very long molecule of DNA
-can be from 2 to 8 cm long yet is packed into about a on micrometer3 volume, obvious tightly folded
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Weight of DNA in human
-each cell contains 6 picograms of DNA
-if stretched into one linear strand it would stretch 1 meter in length.
-chromosomes are DNA-protein associations that allow a thousand-fold condensation of DNA in the nucleus
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Histones
-major quantitative proteins that package the DNA
-H1, H2A, H2B, H3 and H4. Two copies of H2A-H4
-basic proteins due to high levels of arginine and lysine.
-at physiological pH the histones have a large net positive charge and can bind to negatively charged molecules (like the expose phosphates in DNA)
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Nonhistones
-regulatory proteins, enzymes, and other structural proteins
-very heterogenous class of proteins
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Chromatin
-combination of DNA with the histones
-highly condensed=heterochromatin
-less condensed=euchromatin
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Nucleosome
-chromatin contains a repeating unit termed nucleoside.
-nucleosome contains approx. 200 base pairs of DNA, two molecules of histones 2A, 2B, 3, 4 and 1 molecule of histone H1.
-H1 histone is bound to the linker DNA
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Octamer
-core particle of nucleosome
-DNA is wrapped around the exterior of the sphere.
-surface area of a sphere, a large amount of DNA can be packaged into a relatively small area.
-histone H1 proteins interact to form a solenoid structure
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Nucleosome size
-width is ~11nm (100A).
-in chromosome-nucleosomes are packaged upon each their to form a structure which is approximately 30nm (300A) at the primary (solenoid level)
-H1 hist one, through binding to both the DNA of the core particle and the linker DNA, plays an important role in this order of packaging.
-the nonhistone proteins help form a scaffold
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