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DNA Flashcards

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1
Q

What is the chemical nature of nucleic acids?

A

5’ side of the nucleic acid has an extra -CH₂ group between the phosphate group and the sugar ring.
3’ side of the nucleic acid have phosphate group directly bound to the sugar ring. (+ free 3’ -OH group)

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2
Q

Name the 3 components in a nucleotide

A

Nitrogen-containing base (purine or pyrimidine)
Pentose sugar (deoxy/ribose sugar)
Phosphate group

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3
Q

Significant chemical group on purine ring structures (N1, C2 & C6)

A

Adenine:
C2: -H
N1: N double bonded to C6
C6: -NH₂

Guanine:
C2: -NH₂
N1: -NH
C6: C=O

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4
Q

Significant chemical group on pyrimidine ring structures (N3, C4, C5)

A

Cytosine:
N3: N double bonded to C4
C4: -NH₂
C5: -H

Thymine:
N3: -NH
C4: C=O
C5: -CH₃

Uracil
N3: -NH
C4: C=O
C5: -H

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5
Q

Sugar phosphate group provides the backbone of the DNA. How do the ribose sugar ring in A and B form differ from each other?

A

A-form: Exo and endo conformation of C3’

B-form: Exo and endo conformation of C2’

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6
Q

How is nucleoside formed?

A

Coupling of base to sugar via N-glycosidic bond
In purine: N9 of purine formed N-glycosidic bond with C1’ of sugar ring

In pyrimidine: N1 of pyrimidine form N-glycosidic bond with C1 of sugar ring

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7
Q

How is nucleotides formed from nucleosides?

A

Addition of phosphate group to the nucleoside

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8
Q

How are adjacent nucleotides linked together?

A

By phosphodiester bonds between 3’-hydroxyl on the sugar of a nucleotide and the 5’-phosphate group on the sugar of another nucleotide

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9
Q

Are bases planar or non-planar?

A

Bases are planar and any H-bonding interactions with the bases are in the plane of the base

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10
Q

What happens to bases when the pH of the environment fall or rise?

A

When pH increase:
Nitrogen in the bases are deprotonated, losing H atom and hence, losing their base pairings as hydrogen bonds break. This results in the destabilization of DNA double helix

When pH decrease:
Nitrogen in the bases are more protonated, hydrogen bonds between base pairs are broken, destabilizing and breaking the double helix structure.

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11
Q

Bases are capable of undergoing a conversion between ___ forms. A and C can spontaneously isomerize to ___ forms and G, T, U to ___ forms (very ___ frequency of occurrence), which cannot form ___ ___.

A

Bases are capable of undergoing a conversion between tautomeric forms. A and C can spontaneously isomerize to imino forms and G, T, U to enol forms (very low frequency of occurrence), which cannot form base pairs.

*Carcinogens increase the chance tautomeric conversion, destabilizing the DNA

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12
Q

How does spectrophotometry measure nucleic acid concentration?

A

Bases absorb light strongly in the near-ultraviolet region of the spectrum (around 260 nm). This absorption can be used to measure nucleic acid concentration with spectrophotometry.
Using Beer-Lambert Law, A=εCL, to find the concentration.

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13
Q

Synthesis of polynucleotides in vivo (in cell) requires ___ of a ___-energy nucleoside or ___ triphosphate and ___ of a phosphodiester linkage by ___ of water.

*Nucleotide triphosphate = ATP/GTP/CTP/TTP/UTP
- Extremely favorable energetically, used as ___ ___ for a lot of reactions

A

Synthesis of polynucleotides in vivo (in cell) requires hydrolysis of a high-energy nucleoside or deoxynucleoside triphosphate and formation of a phosphodiester linkage by elimination of water.

*Nucleotide triphosphate = ATP/GTP/CTP/TTP/UTP
- Extremely favorable energetically, used as driving force for a lot of reactions

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14
Q

State the 2 important features of all polynucleotides

A
  1. Polynucleotide chain has a sense of directionality:
    One end carries an unreacted 5’ phosphate, the other end an unreacted 3’ hydroxyl group
  2. A polynucleotide chain has individuality:
    The primary structure, determined by the sequence of its bases, is the nucleotide sequence
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15
Q

The default in writing a DNA or RNA sequence is?

A

5’ to 3’

Acceptable compact nomenclatures:
pApCpGpTpT
pACGTT
ACGTT

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16
Q

Explain briefly about Avery and friends’ finding on bacterium Pneumococcus

A

DNA from pathogenic strains of/virulent (bacteria-causing) bacterium Streptococcus pneumoniae could be transferred into nonpathogenic (nonvirulent) strains, making them pathogenic (virulent).

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17
Q

Explain briefly Hershey and Chase study on bacteriophage T2

A

The study showed that when bacteriophage T2 was infected, new genetic instructions appeared in the infected cells. Since only DNA (³²P-labelled) and not proteins (³⁵S-labelled) of T2 entered the infected cell, DNA must carry the new genetic instructions.

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18
Q

What are the secondary and tertiary structure of DNA?

A

2°: Random coil
- Pattern of hydrogen bonding in a polymer

3°: Double helix
- 3D structure as defined by the atomic coordinates
- A vs B vs Z form

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19
Q

Briefly explain about the antiparallel DNA chains

A

The 2 DNA chains are wound around each other, with its bases located on the inside of the helix. The deoxyribose and phosphate are located on the outside, forming the backbone of helix

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20
Q

Explain Chargaff’s rule

A
  • Content of A equals to content of T and content of G equals to content of C
  • Rules can be explained by the Watson-Crick base pairings
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21
Q

Explain the base pairing between different bases

A

Base pair occurs between bases of antiparallel strand forming hydrogen bonds.
A pair with T, stabilized by 2 hydrogen bonds
C pair with G, stabilized by 3 hydrogen bonds

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22
Q

Describe the stacking of base pairs

A

The planes of the bases are perpendicular to the axis of the helix, allowing the stacking of aromatic rings

Base stacking permits strong Van Der Waals interactions between bases, aka stacking interactions, and constitutes the major driving force that stabilizes the double helix

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23
Q

What is the rotation of base pairs called and what is the rotation?

A

Twist

Each base pair is rotated by about 34° with respect to the next, to accommodate to the 10.5 base pairs for each turn in B-form DNA

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24
Q

What is the distance between base pairs called and what is the distance between base pairs?

Study These Flashcards
A

Rise

The rise is about 3.4Å, and given the helical repeat of 10.5 bp/turn, the pitch is 36Å (B-form DNA)

This base pair separation corresponds closely to the Van Der Waals thickness of the bases, so the base pairs are closely stacked within the double helix

25
Two exterior ___ wind between the protruding ___-___backbones, on either side of the base pairs. The grooves are of ___ size, because of how the bases are ___ attached to the sugar. Hence, a ___ groove and a ___ groove
Two exterior grooves wind between the protruding sugar-phosphate backbones, on either side of the base pairs. The grooves are of unequal size, because of how the bases are asymmetrically attached to the sugar. Hence, a major groove and a minor groove *Side facing the sugar and phosphate plane correspond to the minor groove
26
How does complementarity of the 2 strands of DNA help in DNA synthesis
Since A always pairs with T, and G always pairs with C due to complementarity of DNA, DNA synthesis follows the base-pair rule and form 2 exact copies of the original DNA.
27
Explain the mechanism of semiconservative DNA copying
Involves unwinding the strands of a parental DNA duplex, with each strand serving as a template for the synthesis of a new strand, complementary to and wound about each parental strand. Each daughter duplex contains 1 strand of the original duplex and 1 strand of new material Unwinding and replication occurs at the same time, replication of DNA is a fast process
28
Describe the B-form of DNA
- The B-form is seen in DNA fibers prepared in high humidity and is studied by Watson and Crick - Right handed double helix - Structure is generally most relevant for double-stranded DNA
29
Describe the A-form of DNA
- The A-form is seen in DNA fibers prepared in low humidity - Right handed double helix but short and fat - Structure is generally assumed by double stranded RNA and DNA-RNA hybrid molecules
30
Structural difference between A and B forms
1. Position of the base pairs - In B-form: bases lie close to the helix axis (center), which passes between the hydrogen bonds - In A-form: bases lie to the outside, away from the center, and are strongly inclined with respect to the helix axis 2. Shape of double helix - In B-form: major and minor grooves are distinguishable - In A-form: the 2 grooves are more nearly equal in width. The double helix is continuously bent into the major groove, making the major groove compressed and minor groove splayed open
31
Double helix models (like B-form DNA) reflect in a very general way the ___ of DNA in the cell, based on X-ray crystallography of small oligonucleotide fragments Double helix structure involves local variation in: - ___-___conformation - ___/position of a base relative to its ___ partner (base pair structure) - ___/position of a base pair relative to ___ base pair
Double helix models (like B-form DNA) reflect in a very general way the structure of DNA in the cell, based on X-ray crystallography of small oligonucleotide fragments Double helix structure involves local variation in: - Sugar-phosphate conformation - Orientation/position of a base relative to its pairing partner (base pair structure) - Orientation/position of a base pair relative to neighboring base pair
32
Nucleic acid structure is not ___ and varies in response to the ___ sequence and changes by ___ with other molecules Many DNA molecules are ___ as helix axis does not follow a ___ ___. ___ and ___ of bending depends on DNA sequence and is heavily influenced by the ___ of various protein molecules In the nucleus DNA, DNA is packaged with nearly ___ ___ of histone proteins. Thus most DNA are ___ ___ from the ___ of histone proteins into nucleosomes
Nucleic acid structure is not homogeneous and varies in response to the local sequence and changes by interaction with other molecules Many DNA molecules are bent as helix axis does not follow a straight line. Degree and direction of bending depends on DNA sequence and is heavily influenced by the binding of various protein molecules In the nucleus DNA, DNA is packaged with nearly equal mass of histone proteins. Thus most DNA are strongly bent from the wrapping of histone proteins into nucleosomes
33
How does spontaneous deamination of nucleotide bases modify the DNA?
If the deaminated nucleotide base is not repaired, the deaminated base can cause permanent mutations. DNA deamination does not change the size or shape of the bases significantly, but dramatically alters the hydrogen bonding potential, which can cause misinterpretation of the DNA sequence, causing cancer
34
How does depurination events affect the DNA?
DNA experiences a loss of bases through depurination events in aqueous solution Depurination is a result of chemical base modification and spontaneous cleavage of the N-glycosidic bond and there is about 10000 to 100000 depurination sites generated per day per cell
35
What agents cause chemical methylation?
Chemical methylation of DNA is modified by alkylating agents These DNA damaging agents are highly cytotoxic (and carcinogenic)
36
Describe the effect of radiation damage on DNA
Formation of thymine dimer induced by UV light, resulting in the introduction of a kink/bulging upon cyclobutane thymine formation 2 consecutive T in DNA sequence is more prone to dimerising under UV light (increased targeting)
37
Effect of enzymatic methylation of DNA
Methylation regulates gene expression by silencing the gene. Negative effect of methylation: - If gene of enzyme needed to suppress tumor cell is methylated, the enzyme is not produced and hence tumor suppression does not occur, leading to uncontrolled growth of cancer cell
38
Describe the specific enzyme modification system for DNA
Each restriction endonuclease has a cognate recognition sequence of 6 base pairs. When the 6 base pairs are recognised by restriction endonuclease, it cleaves to form sticky/blunt ends. Both cleaved vector and DNA are extracted to form plasmids and ligated
39
Describe the 3 distinct structure of Z-DNA
1. Contains polynucleotides with alternating purine and pyrimidine in each strands are able to form left-handed DNA (Z-DNA) - Alternating pnt: GC, GT, AC, AT repeats 2. Different orientation of the bases - Rotation about the N-glycosidic bond permits various geometric relationships of the base to the sugar - The syn and anti forms refer to 180° opposite conformations of the base to the sugar * Z-DNA requires anti for pyrimidine and syn for purines 3. Function of Z-DNA in vivo appears to be related to the gene regulation and relieving conformational stress in DNA
40
Difference between A, B, and Z forms: Helical sense Diameter Base pairs per helical turn Helix rise per base pair Base tilt normal to the helix axis Sugar pucker conformation Glycosyl bond conformation
A : B : Z Right handed : right handed : left handed ~26Å : ~20Å : ~18Å (narrow) 11 : 10.5 : 12 2.6Å : 3.4Å : 3.7Å 20° : 6° : 7° C-3' endo : C-2' endo : C-2' endo for pyrimidine and C-3' endo for purine anti : anti : anti for pyrimidine, syn for purine
41
How do hairpin loops form in single-stranded nucleic acid molecule?
Single-stranded nucleic acid molecules have self-complementary sequence elements which allows the polymer to fold back on itself and form base-paired structures
42
What are palindromic DNA and cruciform?
Palindromic DNA refers to a segment of duplex DNA in which the base sequences of the 2 strands exhibit 2-fold rotational symmetry about an axis (2 strand can form separate hairpin loops) Cruciform are double hairpins which can be formed with palindromic DNA sequences
43
How are triple helices and Hoogsteen pairing formed?
Triple helices are formed from 1 all-pyrimidine containing strand and 1 all-purine containing strand, where positive charges on cytosine (of the tail end of the double strand) facilitates the formation of the H bonds with the double strand Hoogsteen interaction occurs on the major groove side of A or G and is a non-Watson-Crick hydrogen-bonded motif in nucleic acids where a purine base adopts a syn conformation and interacts with a pyrimidine base, forming triple-helix DNA structures.
44
How are quadruplex DNA formed?
Guanine-rich repeats can form tetraplexes via base-pairing patterns or from a G tetraplex structure, which forms 2 successive tetraplets
45
What are the 2 distinct features of circular DNA?
- Circular DNA do not have free 5' and 3' ends - Molecules may involve either a single stranded or 2 strands intertwined in a double helix (highly stable in nature, used in molecular cloning)
46
Briefly describe the supercoiling of circular DNA
- Supercoiling is a 3° structural feature of DNA as it involves a higher-order folding of elements of regular secondary DNA nature - Supercoiled DNA molecules have twists of the DNA double-helix about itself, with the helix axis crossing over itself one or more times *When the coil coils, it forms a coiled coil, increasing torsion stress results in the formation of supercoils, increasing stress on the double helix
47
What is a topoisomer?
Topoisomers are circular DNA that can assume relaxed and supercoiled forms which differ only in their topology
48
Topoisomer vs topoisomerase
Topoisomers can be interconverted by cutting and resealing the DNA Topoisomerase are enzymes that can cut and reseal DNA
49
Double helical chains of DNA have a remarkable ability to ___ from one another and to ___ again, due to the ___ of the secondary and tertiary structures
Double helical chains of DNA have a remarkable ability to dissociate from one another and to reassociate again, due to the stability of the secondary and tertiary structures
50
Rupture of hydrogen bonds between bases can be due to ___ ___ or ___ of the H+/OH- concentration (___), causing the 2 strands to come apart
Rupture of hydrogen bonds between bases can be due to increasing temperature or alteration of the H+/OH- concentration (pH), causing the 2 strands to come apart
51
Describe the effect of increasing and decreasing pH on DNA
Increasing pH deprotonates the ring nitrogen atoms of guanine and thymine Decreasing pH protonates the ring nitrogen atoms of adenine, guanine, and cytosine Alteration of acidity can cause purine glycosidic bonds to break via hydrolysis, Effect of pH is much larger than effect of temperature as the slight change in pH degrades the RNA in the solution
52
Explain melting and melting temperature (Tm)
Melting: dissociation of the 2 chains with increasing temperature occurs at a defined temperature Tm: temperature at which 50% of the DNA exists in the single-stranded state
53
What does the hyperchromic effect refer to?
Hyperchromic effect: the melting of DNA causing the unstacking of base pairs, which results in increased absorbance at 260nm * Single stranded has higher absorbance than double stranded * An important property used to check for the melting of DNA, by increasing temperature to check for presence of single strands
54
How is Tm related to the base-pair composition of DNA?
Tm is strongly influenced by the base composition of the DNA Tm of DNA increases as the percent (G+C) increases G+C have 3 H bonds, more energy required compared to A+T
55
State the equation to approximate the Tm of DNA-DNA hybrids, RNA-DNA hybrids, and PCR primers
DNA-DNA: Tm = 81.5℃ + 16.6(logM) + 0.41(%GC) - 500/L RNA-DNA: Tm = 79.8℃ + 18.5(logM) + 0.58(%GC) + 11.8(%GC)² - 820/L PCR: Tm = 2℃ (N(A) + N(T)) + 4℃ (N(G) + N(C))
56
In the Tm equation? what does M stand for and how does it affect the Tm?
M is the molarity of monovalent cations Monovalent cations are used to neutralise the negatively charged phosphate groups, reducing the repulsion between the negatively charged phosphate groups, and thus stabilizing the base sequences
57
How does temperature below and above the Tm affect primer binding to DNA?
At Tm, half the primer binds to the DNA, half is not attached Above Tm, more bases of the primer are not attached to the DNA (No effective binding) Below Tm, primer becomes more non-specific, and can bind to non-target regions
58
5 uses of PCR
- Selective amplification of exceedingly small amount of DNA samples in vitro - Molecular cloning: with restriction enzymes - DNA sequencing - Diagnosis: introduce markers and mutations - Forensic analysis of small samples from biological materials like blood, semen, and hair

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