L4: DNA Structure

Question 1

Deoxyribonucleic acid (DNA)

Answer 1

1. nucleosides, nucleotides, and polynucleotide
2. DNA double helix
   
   • 5’ - 3’ polarity
   • anti-parallel nature of double strand
   • complementary structure of double strand
     
     • -> one strand is 5-3 while other is 3-5
3. other important prop:
   
   • denature and renature
   • sequence and sequence homology
   • supercoil

Question 2

4 DNA biochemical properties

Answer 2

1. negatively charged and polar (water loving)
   
   • nucleic acids = hydrophilic due to PO3- along the sugar phosphate backbone
2. precipitated in ethanol/salts, but soluble in water
   
   • Na+ neutralize PO3- groups on nucleic acids –> make molecule less hydrophilic (less soluble in water)
   • ETOH enhances the ability of salt to interact with DNA
3. DNase (deoxyribonuclease) degrades DNA by hydrolysis of phosphodiester bonds
   
   • phosphodiester bond–group of string covalent bonds between a phosphate group and two 5-carbon ring carbohydrates (pentoses) over two ester bonds
4. DNA absorbs UV light (260nm) due to aromatic ring structure in the bases
   
   • dsDNA (double strand) absorbs less at 260nm than ssDNA (single strand) because the bases become unstacked and can absorb more light

Question 3

Nucleoside

Answer 3

sugar + base

Question 4

Nucleotide

Answer 4

sugar + base + phosphate

Question 5

Polarity of DNA strand

Answer 5

• linear polymer or mononucleotides joined by phophodiester bonds bewteen 3’C on one nucleotide and 5’C of the next nucleotide
• bases are all joined to the 1’C
• has polarity 5’-3’

Question 6

DNA Double Helix

Answer 6

• double helix
• right handed
• anti-parallel strands
• strands held together by H-bonding between base pairs on opposite strands and by hydrophobic stacking interactions between base pairs on the same strand

Question 7

Base paris

Answer 7

A = T --> 2 H-bonds
G = C --> 3 H-bonds

Question 8

Base pairing rules

Answer 8

• 2 strands are complementary to each other
  
  • A-T & G-C
  • ex: 5’-TGCCAGT-3’ –> 3’-ACGGTCA-5’

Question 9

DNA strand characteristics

Answer 9

• usually presented as:
  
  • TOP: 5’(left) to 3’(right) –> BOTTOM: 3’(left) to 5’(right)
• size of DNA:
  
  • 1bp = 660 Dalton
  • 1kb (1,000bp) = 660kB
  • 1mb (1,000,000bp) = 6.6 x 10^5 kD

Question 10

DNA can be denatured and renatured

Answer 10

• denature (melt)
  
  • ds DNA –> ss DNA
  • occurs at high temp or lower salt conc or extreme pH
    
    • -> break up H bonds between complementary bases
• renature (anneal)
  
  • ss DNA –> ds DNA
  • occurs at low temp or high salt conc or neutral pH
    
    • -> favors H bond formation

Question 11

Thermal denaturation of DNA

Answer 11

1. occurs at narrow range temp
2. temp corresponding to half the increase in absorption at 260nm–Tm
3. at Tm, 50% DNA is denatured
4. G:C rich DNA in general has higher Tm than A:T rich DNA
5. perfect matched DNA in general has higher Tm than mismatched DNA

Question 12

DNA hyberdization

Answer 12

• annealing of two ssDNA strands
• only occurs when two DNA sequences have homology
  
  • because in mixture of DNAs, only complementary strands will reassociate
• bases of southern blots, FISH, DNA microarrays

Question 13

DNA sequence

Answer 13

• linear order of nucleotides of a DNA strand (5’-3’)

Question 14

DNA sequence homology

Answer 14

• similarity between the sequences of two DNA molecules

Question 15

Flourescence in situ hybridization (FISH)

Answer 15

• can be used to mark regions of DNA

- can be used to detect chromosome abnormalities

Question 16

DNA hybrid formation

Answer 16

ex:
DNA 1: 5'-ATGCT --> 3'-TACGA
DNA 2: 5-CAT -------> 3'-GTA
HYBRIDS: (mix and renature)
                 5'-ATGCT --> 3'-GTA
                 5'-CAT -------> 3'-TACGA

Question 17

Watson-Crick model of DNA structure

Answer 17

1. each DNA molecules has 2 strands of polydeoxyribonucleotide
2. 2 strands spiral around each in antiparallel direction to form a right-handed helix
3. pyrimidine (1 ring bases: T&C) from one stand always form H bonds with purine (2 ring bases: A&G) from other strand
   
   • explains base comp rules
     
     • -> C:G = 3 H bonds A:T = 2 H bonds
4. bases are inside the double helix, sugar-phosphates backbones are outside the helix (backbone)
   
   • each turn of the helix contains 10 base pairs of DNA
5. double helix is held mainly by H bonds between bases from opposite strands
   
   • H bonds can be broken and reformed

Question 18

DNA supercoiling

Answer 18

• when circular DNA molecules is isolated and analyzed using gel electrophoresis, it often migrates as two forms, one migrates faster than the other
• SUPERCOIL–fast-running form of circular DNA that has a more compact structure
  
  • twisted circular DNA
  • very very long linear DNA could also form the supercoiled structure
  • has more or less than 10 bp per turn
  • provides a basis for packaging of DNA in the cell, but it represents a problem in DNA replication

Question 19

Analysis of SV40 DNA by agarose gel electrophoresis

Answer 19

• supercoiled DNA is a more compact molecule

- migrates faster than relaxed DNA

Question 20

DNA content of cells

Answer 20

• DNA of bacterium –> 1000x as long as length of cell and consists of 4x10^6 bp
• DNA of human cell –> 100,000x the cell diameter and consists of 3x10^9 bp

Question 21

Eukaryotic SNA is packed into chromosomes that consists of both DNA and protein

Answer 21

DNA –> nucleosomes –> 30 nm fiber –> interphase chromosome –> metaphase chromosome –> entire chromosome
- nucleosome–contains core histone complex and 150-200 bp DNA

Question 22

Genome

Answer 22

• all genetic information (DNA) for a given species
• HUMAN GENOME–all genetic information on 22 autosomes plus the X&Y chromosome
• most of the genome in higher eukaryotes is made up of sequences that do not code for genes
  
  • in humans, about 95% of genome