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Bioc2201 > Nucleic Acids > Flashcards

Nucleic Acids Flashcards

1
Q

What is the repeating unit of DNA?

A

o A nitrogenous base / base / nucleobase (purine or pyrimidine) linked to
o A sugar (ribose or deoxyribose), which is linked to
o A phosphoric acid (phosphate)

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2
Q

Which are the purine bases? Which are the pyrimidine bases?

A

Purines are Guardian Angels with Two Wings (G, A) - two rings
Pyrimidines are CUT (C, U, T) - one ring

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3
Q

What is the point of attachment of purine and pyridine with sugar in a nucleotide?

A

Nucleoside

C-1’ of the sugar to the N-9 in purine
C-1’ of the sugar to the N-1 in pyrimidines

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4
Q

What is a phosphodiester linkage? What is the acid that reacts to form a phosphodiester linkage?

A

The bonds that connect nucleotides (3’-5’ phosphodiester linkage; 3’-OH on the first sugar joins to the 5’ carbon of the adjoining sugar)

The two phosphoric acid esters on the phosphate that join adjacent nucleotides react to form this linkage.

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5
Q

What is meant by ‘polarity’?

A

The 3’-5’ direction of the strands.

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6
Q

What is meant by the term ‘antiparallel’?

A

One strand runs in the 5’ to 3’ direction, and the other strand runs 5’ to 3’ in the opposite direction.

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7
Q

How DNA formed

A

DNA is synthesised in the 5’ to 3’ direction. Nucleotides units are only added to the 3’ end (and not the 5’ end)

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8
Q

What are the main components of chromatin?

A

DNA is wrapped tightly around histones.

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9
Q

What is the nucleosome?

A

The structural unit of chromatin. Short segments of DNA wrapped around a ‘core’ of 8 histone proteins

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10
Q

What are the two major steps in gene expression?

A

Transcription and translation

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11
Q

Compare nucleotides and nucleic acids

A

Nucleotides are the polymers that form nucleic acids. they are connected by phosphodiester bonds to form nucleic acids (polymers)

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12
Q

Describe the structural differences between the bases.

A

Pyrimidines count from the bottom N.
Purines start anticlockwise from the left N, then clockwise from C-7.

A has an NH2 group on C-6.
G has a double bond O in that position.

C has an NH2 group on C-4
T has a CH3 group on C-5
U has neither of these groups.

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13
Q

What is the correct NUCLEOSIDE NOMENCLATURE for the bases in RNA and DNA?

A

Adenine - Adenosine - deoxyadenosine
Guanine - Guanosine - deoxyguanosine
Cytosine - Cytidine - deoxycytidine
Thymine - thymidine or deoxythymidine (not present in RNA)
Uracil - Uridine (not present in DNA)

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14
Q

What is a phosphoester bond? Which positions on DNA and RNA sugars can be esterified and which is most common?

A

A bond between phosphate and ester. OH, groups, 2’ in RNA and 3’ and 5’ in both can be esterified however 5’ is the most common.

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15
Q

Can nucleosides be esterified to more than one phosphate group? Describe the nomenclature of this.

A

Nucleosides are base-sugar groups, liked via glycosidic linkages.
The number of phosphate groups attached to the 5’ end of the sugar determines the name.

i.e. X amount of phosphate groups bound to adenosine:
1 = adenosine 5’- monophosphate
2 = adenosine 5’-diphosphate

i.e. X amount of phosphate groups bound to a deoxyadenosine:
3. deoxyadenosine 5’- triphosphate

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16
Q

What are some features of DNA structure?
1. How many nucleotides per helical turn?
2. What is the distance between C1’ bonds, and between bases?
3. What stabilises DNA?
4. Why does DNA have hydrophobic/philic areas?
5. Where are the major and minor grooves?
6. Compare B, Z and H-DNA. What is it and where is it found?

A
  1. 10 nucleotides per helical turn.
  2. 1.08nm between C1’ bonded pairs and 0.34nm between each base up a strand.
  3. H-bonding and van der Waals forces
  4. Hydrophobic core prevents water from disrupting the bases whereas the hydrophilic outer allows DNA to be soluble in aqueous environments
  5. closer strands create a minor groove and the strands further apart create a major groove, this is where proteins/ drugs interact.
    • B-DNA is normal right-handed helical DNA that turns clockwise.
    • Z-DNA is the alternate left-handed form with 12 bases per turn, found in genetic disease but mainly occurs naturally and has a biological function.
    • H-DNA has triple-helices and is formed by trinucleotide repeats. Found in genetic diseases.
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17
Q
  1. How is DNA stored in the nucleus?
  2. How do histones neutralises DNA?
  3. How do histones orginise DNA?
  4. Why are nucleosomes called an octomer?
    5 .What is a linker H1?
A
  1. In the form of chromatin
  2. DNA is negatively charged due to phosphate. Histones have high proportions of positively charges Arginine and Lysine amino acids which neutralise the backbone of DNA.
  3. Histones organise DNA into nucleosomes (the structural unit of chromatin). DNA strands wrap around the histone core resulting in reduced length.
  4. Because it’s made up of 2 histone molecules each of H2a, H2B, H3 and H4
  5. H1 binds to the outside of the core particle and to the linker region. it is involved in chromatin condensation.
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18
Q

What is the function of DNA as genetic material?

A

DNA replication and transcription and translation

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19
Q

What is DNA replication?

A
  • ensures daughter chromosomes are identical
  • complementary base pairing NOT identical
  • one strand acts as the template
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20
Q

What is Gene expression?

A

genetic code being transcribed into mrna and then translated into a protein via protein synthesis

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21
Q

What are the type of rna?

A

mRNA (5%)
tRNA (15%)
rRNA (80%)

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22
Q

What is RNA primary and secondary structures?

A

Primary - single-stranded
Secondary - ‘stem-loop’ structure. Stem fold back on itself, the loop is a region with no complementary base pairing

23
Q

What is mRNA?

A
  • exact single strand copy of DNA (U replaces T)
  • RNA polymerase transcribes
  • template for protein synthesis
24
Q

What is tRNA?

A
  • carries amino acids to ribosomes as directed by codons in mRNA. Anticodon is the recognition site on the tRNA.
  • 2D structure forms cloverleaf.
  • 3D shape, L shaped.
25
Q

What is rRNA?

A
  • Part of the ribosome which binds mRNA to tRNA
  • secondary stem-loop structure
  • contains 2 subunits, small decodes genetic message, large catalyses peptide bond formation
26
Q

Compare base paring between DNA and RNA.

A

DNA - intermolecular. bases from one dna strand pair with bases on the opposite strand

RNA - intramolecular. bases pair when RNA folds in on itself

27
Q

Chemical properties of genetic material at low pH (below 1)

A
  • breakage of phosphodiester linkages
  • breakage of glycosidic links
  • H bonds break
28
Q

Chemical properties of genetic material in weak acid (pH 2-4)

A
  • depurination
  • removal of purine bases
  • the glycosidic link between purine and sugar is broken
  • phosphodiester links remain intact
  • glycosidic link on pyrimidines more stable
29
Q

Chemical properties of genetic material in high pH (pH 10-13)

A
  • Unstable RNA
  • 2’ OH groups break phosphodiester links
  • DNA has no 2’ OH group and is relatively stable
  • although causes dsDNA to become ssDNA
30
Q

What wavelength does genetic material absorb UV light? What is the hyperchromic shift phenomenon?

A
  • ssDNA aborbs 40% more UV than dsDNA at 260nm
  • hyperchromic shift refers to UV absorbance increasing when dsDNA is denatured into ssDNA
31
Q

How does temperature affect DNA? What is Tm?

A
  • dsDNA denatures at approx 90 degrees into ssDNA.
  • Tm is the melting point at which there is 50% ds and50% ss. It depends on the particular DNA sequence
32
Q

What is the relationship between Tm and GC content?

A
  • A-T base pairs have 2 H bonds
  • G-C has 3
  • DNA with high % GC is more stable
  • therefore more energy (heat) requires to separate
33
Q

What is shearing?

A
  • Mechanical breakage of DNA
34
Q

What barriers do we need to get through to extract DNA and what don’t we want in our sample?

A
  • cell membrane and nuclear envelope
  • proteins and rna
35
Q

What are the 5 steps in DNA extraction?

A

DNA lysis, purification, precipitation, wash dna pellet, rehydration

36
Q
  1. What does Cell lysis mean?
  2. What is the solution we use?
  3. What does SDS do to the cell?
  4. What do we do to the DNA sample after adding SDS?
  5. What does heat do to the sample?
A
  1. to break open
  2. Sodium Dodecyl Sulfate
  3. Disrupts cell membrane and removes proteins
  4. Heat to 65 degrees for 20 minutes
  5. This denatures any proteins, which have an optimal temperature of 37 degrees
37
Q

What is phenol/chloroform extraction?
Compare is to protein precipitation.
What does it remove?
what are the layers/ phases formed after centrifuge?

A

A form of DNA purification.
Yields higher purity DNA but requires harsh/ harmful chemicals
removes proteins lipids and cell debris
Formes top DNA supernatant (aqueous), interphase has proteins and bottom layer is phenol/ chloroform (organic layer)

38
Q

What is protein precipitation?
What are the steps involved?
How does is work?

A

A form of DNA purification.
Add solution to DNA sample, centrifuge, and transfer supernatant to a new tube.
The high salt buffer of the protein precipitation solution decreases the solubility of the proteins.

39
Q

What is DNA precipitation? What are its 2 methods?

A

Chemicals are added that cause DNA to form an insoluble solid (precipitate).
Isopropanol precipitation uses isopropanol and glycogen, Ethanol precipitation uses sodium acetate and 100% ethanol.

40
Q

What do we add and why in isopropanol precipitation?

A

Isopropanol - DNA cannot dissolve in alcohols, this precipitates DNA from the solution.
Glycogen - acts as a co-precipitant; also cannot dissolve in IPA. Binds to DNA and traps it

41
Q

What do we add and why in ethanol precipitation?

A

Sodium acetate is a salt that dissociates Na+ that neutralises negatively charge phosphate of DNA, making it less soluble in water.

DNA is insoluble in ethanol. This precipitates DNA from the sample solution.

42
Q

How is the DNA pellet washed? Why don’t we use propanol?

A

with 70% Ethanol, which will further precipitate the pellet and 30% water that should dissolve any left-over salts. Propanol is less volatile and would require more time to airdry.

43
Q

Why do we rehydrate the DNA pellet and what is used?

A

Nucleic acids will dry up if left as a pellet. The hydration solution will depend on what the DNA will be used for in downstream experiments.

44
Q

What is the A260/A280 ratio?

A

This ratio compares the nucleotide absorbance at 260 and protein absorbance at 280, it allows us to determine the purity of DNA. A ratio between 1.8-2 is considered pure.

45
Q

What are 3 advantages of a nanodrop spectrophotometer?

A
  • determine the amount of DNA, RNA or protein in sample
  • calculate A280/A260 ratios
  • efficient
46
Q

What are the 3 steps in agarose gel electrophoresis?

A
  1. load DNA/RNA sample into agarose gel
  2. Apply current (electrophorese), genetic material is negatively charges and migrate to the positive electrode
  3. Gel is imaged under UV light
47
Q

Why are nuclei acids visualised using intercalating dyes? What are 3 ways dye can be added?

A

The dye causes nucleic acids to fluoresce under UV light.

  1. In agarose gell mix (pre-cast)
  2. Included in gel loading buffer
  3. Gel can be immersed in a dye solution at the end (post-electrophoresis gel staining)
48
Q

How does DNA move through gel electrophoresis?

Compare how large/ bulky move compared to smaller/compact fragments.

How is the size of DNA/ RNA determined?

A
  • Agarose gel has microscopic pores that act as a sieve to allow fragments to move and separate when the electrode is applied
  • smaller/ compact fragments move further and faster
  • using a comparison ladder of know sizes
49
Q

Compare the genes and bp in the following and explain why Arabidopsis has more genes:

Human DNA
E. coli DNA
Arabidopsis (rockcress plant)

A

Human: 3 billion bp, 25,000 genes
E.coli: 4.6 million bp, 4,400 genes
Arabidopsis: 125 million bp, 25,498 genes. Has more genes than humans but humans have more DNA.

50
Q

What are nucleases?
Draw a table comparing the different types.

A

Enzymes that cut nucleotides.

Exo cut one at a time from the ends of DNA. Can only act in one direction.

Endo are site-specific and act within DNA by recognising particular sequences.
e.g. Restriction enzymes

51
Q

How were restriction enzymes discovered?

Explain restriction enzyme nomenclature, use examples:
- 3rd RE Haemophilus Aegyptus
- 1st RE Escherichia coli strain R

What is restriction enzyme digestion?

A

Discovered originally in bacteria that naturally use these enzymes as a defence mechanism

Identified by a name (bacteria it was discovered in) and a number (order it was discovered in).
Haelll and EcoRl

When one or more REs are added to a DNA sample to digest it. They will cleave when they encounter the recognition sequence.

52
Q
  1. What is a cleave site?
  2. What is the recognition sequence in RE?
  3. What are palindromes?
  4. What is an overhanging/ sticky/ cohesive cut? Provide example.
  5. What is a blunt end? Provide example.
A
  1. Site of cutting
  2. Sequence it cleaves off
  3. Sequences of DNA that read the same forward and backward, that is the recognition sequence is the same when read in the opposite direction.
  4. Asymetrical cut, e.g. BamHl
  5. Symmetrical cut, e.g. Haelll
53
Q

What are cleavage frequencies and how are they calculated? What is the exception?

A

Used to predict how often a RE will cleave a random sequence of DNA.
= probability of a specific sequence occurring in a random sequence.

1 / 4^n

(n being the number of known bases in the sequence)

An exception is when a base is unknown and is written at N, in this case, it is not included when counting n.

Bioc2201 (8 decks)

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