BIOCHEM LAB - Nucleic Acids Flashcards
monomer unit of nucleic acids
nucleotides
NA carries a __ charge on the oxygen of the phosphate component
-
not nutrient biomolecules because —
they can be biosynthesized
NA are branched or unbranched?
unbranched
a 5-carbon monosaccharide in ring form as heterocyclic hemiacetal derived from furan
PENTOSE
has a deoxyribose sigar with OH group on 3’ position of sigar ring
DNA
has a ribose sugar with OH group at 2’ and 3’ positions of sugar ring
RNA
No NA, NO ___
Organism
blueprint of an organism is encoded in
NA
nitrogen base is attached to the 1’ carbon of sugar through a what type of bond
glycosidic
fused bicyclic base
purine
monocyclic base, 6membered rin
pyrimidine
purines are attached to sugar by what beta-glycosidic bond
N9 - C1
pyrimidines are attached to sugar by what beta-glycosidic bond
N1-C1
More susceptible to H hydrolysis due to greater dipositivity of glycosyl bonds
glycosidic bonds to purine
IUPAC name of cytosine
2-oxo-4-aminopyrimidine
IUPAC name of thymine
5-methyl-2,4-dioxopyrimidine
IUPAC name of uracil
2,4-dioxopyrimidine
bases that are planar
pyrimidine
bases that are very nearly planar with a slight pucker
purine
IUPAC name of adenine
6-aminopurine
IUPAC name of guanine
2-amino-6-oxopurine
most polar base
Guanine
least polar base
thymine
gives NA their acidic and anionic character
phosphate
phosphate is attached to what carbon atom
5’ c of the sugar component
type of bond between phosphate groups
phosphate anhydride or pyrophosphate bond
small proteins containing basic amino acids: lysine and arginine
histones
in circular DNA, the type of hydrogen bonding is intra or inter
INTRA
forms the solenoid supertstructure
DNA-histone complex
DNA-histone complex forms ____ structure when it undergoes coiling
solenoid supertstructure
Chromatinf iber is formed by
solenoid interaction with scaffold protein
undergoes compaction by folding and coiling
short-lived NA
synthesized as the need arises
RNA
mostly found in cytoplasm with non-histone proteins in ribosome
RNA
found in nucleolus of nucleus where ribsomes are synthesized
rna
WHERE THE DNA IS ALSO FOUND
in mitochondria and chloroplast
responsible for the 3d structure of DNA
James Watson and Francis Crick
xray diffraction data
Maurice Wilkins
Rosalind Franklin
double-helical forms by which DNA exists
A B C D E H Z L
natural DNA
most stable and dominant under physiological conditions
B-DNA
handedness of B-DNA
right handed (2 helix)
pitch of B-DNA
3.4 nm
rise of B-DNA
0.34 nm
helical diamtere of of B-DNA
2.0 nm
number of base pair per turn of of B-DNA
10
has minor and major grooves and is found in the cells
B-DNA
coiling od B-DNA is described as
plectonemic
this NA may contain local regions of short complementary base-pairing
RNA
NA that has a strong tendency to adopt a random coil conformation
RNA
NA found in RER
RNA
CHARGAFF’S RULES
Base composition of DNA ___ from one species to another
varies
CHARGAFF’S RULES
DNA of closely related species have ___ base composition
similar
CHARGAFF’S RULES
DNA of diff tissues of an organism have ___ base composition
same
CHARGAFF’S RULES
base composition of DNA (change or does not?) with age, nutritional, state etc
NOT CHANGE
CHARGAFF’S RULES
the ratios of adenine to T and G-C were nearly
1.0
CHARGAFF’S RULES
total number of purines in one strand is ____ to total number of pyrimides in complementary strand
same
structure which descirbes nucleotide base sequence
polarity of sugar backbone
primary
by convention, sequences are written in what direction
5’ to 3’
stabilizing factos in primary structure
covalent bonds
tautomerism
resonance energy
it is a phosphodiester bond bet nucleotide monomers
the main stabilizing factor
covalent bonds
covalent PHOSPHODIESTER bonds are stable between what ph
pH 3 AND 12
at extremes in pH, phosphodiester bonds are ___
HYDROLYZED resulting in scission or shearing of DNA
stabilizing factor of primary struc that is pH dependent
tautomerism
a stabilizing factor formed when nitrogen bases are stabilized by electron delocalization
tautomers
what is formed when nitrogen bases are stabilized by electron delocalization
tautomers
type of tautomer that is predominant and more stable
commone
N bases are aromatic, which means they are
thermodynamically stable
the higher resonance energy, __
more thermodynamically stable
which type of base is more thermodynamically stable
purines
degree or thermodynamic stability
A>G>C>U>T
structure which descirbes the helical conformation of NA
secondary
conformation that is more thermodynamicallys table
helical instead of random coil
conseuqence of RNA being SS with regard t degree of secondary structure
lesser in degree than DNA
stabilizing factors in secondary structure
complementary base-pairing via intermolecular HB in DNA or intra in RNA
base stacking via pi-pi complexation rxns
T or F
E delocalization becomes more extensive when complementary bases are HB to each other
T
Properties of DNA related to 2ndary structure
DNA is relatively stable but not inert. Why
strands separate during repli/transcrip
Properties of DNA related to 2ndary structure
DNA solutions are viscous because
- rigidity of double helix
2. high length/diameter ratio
Properties of DNA related to 2ndary structure
DNA is most stable and soluble in salt solutions with ___ high or low conc
HIGH
salt solutions of less than ___ M would weaken HB
0.1 M
effect caused by exposure of nitrogen bases
hyperchromic effect
effect of hyperchromic effect to absorbance
increase in 260nm
denaturation is reversible or irreversible?
reversible
hyperchroic effect is caused by the
exposure of nitrogen bases caused by unstacking caused by Hbonds disruption caused by unwinding
conformation of SS DNA after hyperchromic effect
random coil
process by which denatured complementary strands of DNA can reform a duplex DNA structure
renaturation or annealing
as DNA anneals, its absorbance will ______
decrease - causing hypochromic effect
relationship of rate of renaturation to concentration of complementary strands
direct
when does dna become less viscous
high temp
treatment with alkali
temp at which dna dissociate or unwinds
80-90
temp related denaturation
melting
temp at which 50% of double helix is unwound
melting pt or transition temp
Tm is determined using a
thermal denaturation curve
temperature profile
melting curve
temp at which dna renatures
below Tm
the more GC pairs, the ___ melting point
higher
the more GC pairs, the ___ melting point
lower
structure which describes degree of supercoiling
tertiary
level of structure which describescovalent modification of RNA
tertiary
NA makes up how many percent of cell’s dry weight
5-15
what type of organs are rich in NA
highly cellular
spleen liver thymus pancreas
what type of organs are poor in NA
brain muscle tissues
choice sample for DNA
tissues with cells with high nuc to cytoplasm volume ratio
very good source of dna
lymphoid tissue
best sourceof dna
thymus
good substitute for source of dna
spleen
types of interactions responsible for the rigid molecular configuration of NA
phosphodiester bonds
HB
VDW forces
interactions involved in complementary base-pairing via HB inDNA and RNA
phosphodiester bonds
HB
factors for choosing material for experiments
high content of DNA RNA
low DNase and RNase activity which are both hydrolases
NA from source are readily obtainable in essentially pure form
HB bonding bet complementary base strans is stable between what pH
4 and 10
N,C glycosidic bonds to purine bases are hydrolyzed at pH
3 OR LESS (APURINIC SITES)
phosphodiester bonds and N,C glycosidic bonds are stable up to what temp
100C
condition in which cell wall releases DNA when tissue is homogenized
lysis or disruption
enzyme that causes most serious damage or degradation of DNA
DNAses or deoxyribonucleases
enzymes that catalyze hydrolysis of 3’,5’ phosphodiester linkages
DNAses or deoxyribonucleases
enzyme present on fingertips
causes spurious degradation of NA during purification
nucleases
in the absence of required ___ and in the __, degradative enzyme becomes nonfunctional
ion and in the cold
used to avoid degradation of dna
glass
rubber
plastic tools and ocntainers
native dna is present in cell as what type of complex
DNA-protein complexes
During the extraction process, proteins must be ____from DNA
dissoaciated or separated from DNA
examples of mechanical stress that may cause cleavage shearing or scission of DNA chains
grinding, shaking, stirring, squiritng sol’n through narrown orifices
does not cause damage to secondary structure of dna but reduces length of molecules
mechanical stress
purified dna is best kept in solution or dry
in solution
if precipitated DNA is kept dry, it tends to undergo
denaturation
how to maintain dna in its secondary structure
keep it in 0.1 M acetate buffer
what to do to inhibit growth of microorganism after purifying dna
add 0.001M sodium azide
storage temp must be 4C
RNA of low MW is best kept dry or slurry
dry
RNA of high MW is best kept dry or slurry
slurry under 75% aqueous alcohol
with 2% sodium azide at 4C
scientific name of onion
Allium cepa
steps in dna isolation
homogenization
dissociation and denaturation
purification of NA
steps in dna isolation
disruption of cell membrane and membrane of subcellular particles to release nucleoprotein
homogenization
steps in dna isolation
separates protein (deproteinization) and releases NA
NA is then precipitated out
dissociation and denaturation
steps in dna isolation
based on principles of MW, size, structure, base composition etc
purification of NA
homogenization involves what process to inactivate nucleses
denaturation
onion sample is treated at what temp with a homogenizing solution
60C
homogenizing solution
dilute saline solution
to precipitate nucleoproteins and separate it from other proteins
0.15M
homogenizing solution
breaks ionic interaction between protein and NA and binds to the protein by hydrophobic interaction
makes it negatively charged
5% SDS
homogenizing solution
binds Ca and Mg ions needed to activate nucleases and RNAse and which can form salts with anionic phosphate groups of NA
0.15M sodium citrate
homogenizing solution
to dissolve proteins and bind Ca and Mg ions
0.001M EDTA
WHY heat the onion sample at 60C
to dissolve NA separated from the protein without affecting HB in both NA and potein
why cool in an ice bath
slow down activity of nucleases
why treat with papain or meat tenderizer (dna)
denature all protein in aqueous medium
what does denaturation do with the hydrophobic portion of proteins
expose them to be precipitated out
why add ice cold 95% ethanol
to precipitate DNA and RNA
after isolation describe dna
white fibrous or threadlike ppt which can be collected by spooling onto a glass rod
scientific name of yeast
saccharomyces cerevisiae
how much RNA does yeast contain
4% by weight
in rna isolation, why heat with dilute alkali
separate from protein
extract rna and water soluble proteins
inactivate nucleases
in rna isolation, why mix ethanol with conc HCl
to make alcohol more polar, thus precipitating the less polar NA, RNA
buffer or solution used
tris-EDTA buffer of SSC solution
meaning of TE and SSC
standard saline citrate
tris-EDTA
why does dna and rna have sufficient absorptionin uv range
presence of aromatic nitrogen bases which contain conjugated double bonds
major absorption band for purified dna and rna peaks at about
260nm
primary contaminant in dna and rna
protein material with trp phe and tyr
peak absorbance of protein material with trp phe and tyr
280 nm
TYPICAL absorbance ratio for good quality NA
1.8-2
absorbance ratio for isolated dna
1.8
what does a small ratio indicate
increased contamination by protein
what does a bigger ratio indicate
increased contamination by rna or denatured dna
amt of NA and protein in sol’n can be determined using
monograph
RNA has higher or lower absorption at 260 nm than DNA
higher
why does RNA have higher absorption at 260 nm than DNA
because it is SS, nitrogen bases are exposed
A ratio of highly purified RNA
1.85-2
absorbance of RNA if ti has contaminants such as phenol, polysaccharides, peptides
230nm
The symmetry of the sugar-phosphate backbone imparts a —- to the nucleic acid
Polarity
The single-stranded nucleic acids may contain —– regions of —-
local regions of short complementary base pairing
Since NA is single stranded, it has a strong tendency to adopt what conformation?
Random coil
Location of RNA
Cytoplasm, rER, nucleolus
Thermodynamically stable means lower or higher free energy?
Lower
Intermolecular forces present in pi-pi complexation rxn
Van der Waals
Intermolecular interaction
Why was hydrolysis performed
To separate the components of nucleic acids isolates
