DNA Chemistry and Analysis Flashcards
What are the 4 components of a nucleotide?
- nitrogenous base (purine or pyrimidine)
- sugar (ribose or deoxyribose)
- phosphate
- rare or minor bases
Methylation is an important modification to a major base because ___ ___ can be controlled by this modification
gene expression
Transfer RNA contains a high percentage of ___ bases
minor
Ribosomal RNA contains a high percentage of ____ bases
methylated
What is the most commonly modified base in DNA?
5-methylcytosine
In regards to sugar, DNA contains ____ and RNA contains ____
B-2-deoxyribose; B-D-ribose
DNA contains thymine while RNA contains ____ in place of it
Uracil
In regards to linkage, mononucleotides are joined together by ____ bridges between the 5’hydroxyl group of one nucleotide and the 3’hydroxyl group of the adjacent nucleotide.
phosphodiester
In regards to linkage, the bases are not directly attached covalently to one another. True or false?
true
Nucleic acid sequences are always written from left to right: ___’ to ___’ direction
5;3
The products of strong acid hydrolysis of DNA or RNA are what?
pyrimidine nucleosides, deoxyribose or ribose and phosphate
Base hydrolysis of ____ proceeds through a 2’,3’-cyclic phosphate intermediate
RNA
Since DNA doesn’t contain a 2’ hydroxyl group, it is resistant to ____ hydrolysis.
base
note: this is important method for separating DNA from RNA
DNA is stable in base and RNA is not stable. True or false?
true
What is the product of base hydrolysis of RNA?
2’ and 3’ nucleoside monophosphates
In both prokaryotic and eukaryotic cells, DNA exists as a double helix in which two DNA strands are wrapped around the same axis. True or false?
true
In regards to the double helix, the phosphodiester bond and the deoxyribose moieties that they link from the ____ of each strand with the nitrogenous base projecting into the interior of the double helix
backbone
In regards to the double helix, ____ project to the outside of the double helix where they interact with proteins
phosphates
note: negatively charged phosphates are complexed with cations such as Na, K and Ca - leads to helix stabilization
In regards to the double helix, the nitrogenous bases are stacked in the interior of the double helix with the planes of the bases being parallel to each other and perpendicular to the long axis of the double helix. Because of the proximity of the stacked bases ___ ___ ___ attractive forces contribute greatly to the stability of the helix
van der waals
In regards to the double helix, bases of one strand form ___ bonds with the bases of the other strand such that ___ always pairs with ____
hydrogen; adenine; thymine (and guanine with cytosine)
In any DNA molecule, the amounts of adenine and thymine are always the same while the amounts of guanine and cytosine are always the same. True or false?
true
Hydrogen bonding between Adenine and Thymine is ____ than the bonding between Guanine and Cytosine
weaker (double bond in AT vs triple bond in GC)
What is the distance of a complete turn of the double helix? How many base pairs are needed for one complete turn?
- 4 nm; 10 base pairs
note: this is the B form and it is the most abundant
The grooves in the DNA provide surfaces to which regulatory proteins can bind. The major groove is almost ___ as wide as the minor groove
twice (22 A vs 12 A)
What are the 4 reasons why the double helix is stable?
- there are hydrophobic and electronic interactions between the stacked bases of each strand
- the hydrogen bonds that are formed between the complementary bases of the two strands
- the hydrophobic nitrogenous bases are shielded from the aqueous environment
- the negatively charged phosphate groups and polar sugar moieties are exposed to the aqueous environment
Segments of DNA rich in AT pairs melt at a ____ temperature than GC pairs.
lower
What is the hypochromic effect?
Melting of DNA in solution can be followed by an increase in ultraviolet light absorbance when DNA is heated to its melting temperature. The stacked bases in double stranded DNA decreases the UV absorption relative to free nucleotides in solution
Hydrogen bond formation allow double strand DNA to form two complementary single strands. This slow step is called what? hint: opposite of melting or denaturing
annealing
The most common conformation of DNA (and the one discovered by Watson and Crick) is what?
B-DNA
DNA C has ___ base pairs per complete turn of the double helix with the distance of one complete turn being ___ nm
9; 3.3
note: not thought to occur in vivo
DNA A has ___ base pairs per complete turn of the double helix with the distance of one complete turn being ___ nm
11; 2.8
The nitrogenous bases in DNA A are not perpendicular to the long axis of the double helix but have a tilt of about ___ degrees
20
DNA A froms under low ___ conditions and probably occurs in very ___ stretches in DNA and is sequence dependent
hydration; short
DNA Z has ___ pairs per complete turn of the double helix. DNA Z has been observed in crystals of synthetic hexanucleotide d and in fibers of the alternating d(GC)n polymers
12
Z DNA is in the form of a ___-handed helix who B DNA is in the form of a ___-handed helix
left; right
The deoxyribose-phosphate back bone of __ DNA has a staggered zigzag course.
Z
Within cells, most DNA is in the B conformation although, regions rich in - base pairs may assume the Z conformation
GC
Methylation of deoxycytidine groups in the GC rich regions of DNA may favor ___ DNA formation
Z
True or false? It is impossible for a B-Z transition to occur within a segment of DNA without totally separating the two strands
false; it’s possible
Although not a separate conformation of DNA, it is known that short stretches of ___ residues can cause bending of the DNA helix.
adenine
Six adenines in a row can cause a bend of ___ degrees
18
The expression of genes is generally ___ in regions of DNA with an A-bending sequence
blocked
The nucleotide sequence in the ___ strand is the same as that of the RNA made from the DNA, except uracil will substitute for thymine in the RNA
coding
This 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 to this strand but not to the coding strand
template
What are three factors that can cause denaturation of DNA?
high temperature, high pH, low ionic strength
note: low ionic strength is not sufficient to bring about complete denaturation. however, low ionic strength is combination with high pH or high temp is experimentally used to cause denaturation of double stranded DNA
Does denaturation require energy expenditure to occur in vivo?
yes
The property of self-association of complementary strands has provided the basis of the development of a technique termed _____
hybridization
In his type of blotting, a DNA sample is electrophoresed, denatured, transferred to nitrocellulose and hybridized with a radio labeled probe. This technique is often used to see gene structure or gene complexity.
Southern blotting
In this type of blotting, an RNA sample is electrophoresed, transferred to a membrane and immobilized, and then hybridized with a DNA probe. This technique is used to measure RNA message levels for genes.
Northern blotting
In this type of blotting, protein sample is electrophoresed, transferred to a membrane and then visualized by an immunological procedure
Western blotting
A 5’ exonuclease must be present with a 5’ ___ on the terminal nucleotide to remove it from the polynucleotide chain
hydroxyl
A 3’ exonuclease must be present with a free 3’ OH on the terminal nucleotide to remove it from the chain. What is an example of a 3’ exonuclease?
DNA polymerase I
these are enzymes which hydrolyze in the interior of a polynucleotide. DNase I is an example which cuts in a nonspecific fashion.
endonuclease
____ enzymes are endonuclease that recognize base sequences in double helical DNA and cleave both strands of the duplex DNA.
Restriction
note: they are found in a wide variety of bacteria and restrict growth of bacterial viruses
____ is a phrase which reads the same way forwards and backwards. “madam i’m adam”
palindrome
Restriction enzymes are never palindromic. True or false
false, most are palindromic
_____ DNA molecules are created by linking restriction fragments of DNA from one organism with another. Generally the DNA fragments of from the organism of interest are linked to a bacterial ____
recombinant; vector
Describe the process of cloning a human gene into bacteria
a fragment of DNA obtained from genomic or cDNA library is introduced into a plasmid. The plasmid is reintroduced into a bacteria (transformation). Bacteria processing the “engineered plasmid” are identified and isolated.
What are 3 desirable vector characteristics?
- multiple enzyme choices
- bears genes that code for proteins that impart resistance to antibiotics - permits selection of bacteria
- possesses promoter sequences which permit the insert to be transcribed
Through hydrogen bonding, complementary extensions from enemy digestion facilitate joining of DNA fragments by _____
ligation
Because the size of the piece of foreign DNA that can be inserted into plasmids is mall, other vectors have been developed. These consist of ____ ___ vectors
lambda phage
_____ are plasmids which contain DNA sequences that allow DNA to be packaged into a bacteriophage protein coat. This allows efficient expression of the library.
COSmids
This DNA appears to be localized in the centromere region of the chromosome and probably participates in alignment of chromosomes during mitosis and meiosis. These sequences do not appear to be transcribed. Also referred to as satellite DNA.
highly repetitive
The Alu I family is an example of what type of DNA
highly repetitive
This type of DNA consists of both transcribed and untranscribed sequences.
moderately repetitive
Haploid human cells contain approximately ____ base pairs of DNA
3.5 x 10^9
About ____ genes are estimated to exist for humans
25,000
In eukaryotic cells, the DNA appears in structures called _____
chromosomes
Each cell in the human body contains ___ picograms of DNA. If all this DNA were stretched into one linear strand it would stretch ___ m in length
6; 1
____ are DNA-protein associations that allow a thousand-fold condensation of DNA in the nucleus
chromosomes
These proteins package DNA. There are 5 types.
histone
All the histones are very basic proteins due to the presence of high levels of ___ and _____
arginine; lysine
At physiological pH, the histones have a large net ___ charge and can bind to ___ charged molecules
positively; negatively
The combination of DNA with the histones forms a structure termed ____
chromatin
Chromatin contains a repeating unit termed a ____. Each one contains approximately ___ pairs of DNA, ____ molecules of Histones 2A, 2B, 3, 4, and ___ molecule of histone H1
nucleosome; 200; 2; 1
In the core particle, eight histone molecules form an octamer forming a sphere. The DNA is wrapped around the exterior of the sphere. What is the advantage of this?
a large amount of DNA can be packaged into a small area
Histone H1 proteins interact to form a ____ structure
solenoid
the width of a nucleosome is approximately ___ nm
11 (100 A)
In a chromosome, the nucleosomes are packaged upon each other to form a structure which is approximately ___ nm at the primary (solenoid) level
30 nm (300 A)
The H1 histone, though binding to both the DNA of the ____ particle and the ___ DNA, plays an important role in packaging while the nonhistone proteins help form a ___
core; linker; scaffold
In order for DNA to be replicated and transcribed, the binding of histones to DNA must be disrupted. ____ and ____ of N-terminal amino acids in histones alters the histone charge from + to _
acetylation; phosphorylation
____ of histones occurs strongly in expressed regions of chromatin (gives rise to R-banding of chromosomes in histology)
acetylation
Proteins known as ___-___ group proteins are required for unwinding of nucleosomes to produce regions of DNA that can be transcribed
high-mobility
