Forces That Destabilize the Double Helix

Question 1

The Two Forces

Answer 1

• heat
• negative charge on phosphate in backbone
• affect kinetics of dissociation and reassociation of DNA molecules - basis of experiments in molecular genetics.

Question 2

heat

Answer 2

thermal energy increases the mobility of molecules tending to denature (unwind) the two DNA strands.

Question 3

negative charges on phosphate in backbone

Answer 3

• the repulsive forces of the negatively charged phosphates on the DNA backbone tend to push the two DNA strands apart.

Question 4

DNA absorbance spectrum

Answer 4

• made in a spectrophotometer by passing light of different wavelengths through a solution of DNA and measuring absorbance
• relative max absorbance at 260 nm
• relative min absorbance at 230 nm

Question 5

hyperchromicity

Answer 5

• as double stranded DNA denatures
• 40% increase in A260
• further absorbance if single-stranded DNA broken into nucleotides - residual hyperchromicity

Question 6

From ssDNA to dsDNA

Answer 6

Divide by 1.4

Question 7

Tm

Answer 7

• thermal denaturation midpoint
• point at which DNA is 1/2 double stranded and 1/2 single stranded
• referred to as melting temp

Question 8

from Tm to ssDNA

Answer 8

1.4/1.2 Tm

Question 9

Effect of GC and Cations

Answer 9

• the higher the GC content, the higher the TM. Higher GC content makes a more stable helix and it takes more energy to disrupt the helix.
• the higher the monovalent cation concentration (Na+) the higher the Tm

Question 10

Why to Effect of GC and Cations

Answer 10

• the Tm of GC rich DNA is higher primarily because the HYDROPHOBIC STACKING INTERACTIONS ARE GREATER in GC rich DNA and secondarily because the HYDROGEN BONDS are stronger in GC pairs than in AT pairs
• Salt stabilizes the double helix because the cations shield (or buffer) the repulsive forces of the negatively charged phosphates - forces that tend to destabilize the helix.

Question 11

higher the salt

Answer 11

• the more stable the interaction

- more heat to denature

Question 12

NaCl, GC, Stability, and TM

Answer 12

• if NaCl increases
• and/or GC increases,
• Stability increases
• and TM increases
• more energy to melt double helix.

Question 13

Nucleic Acid Hybridization

Answer 13

• Effects of GC content and salt concentration have implication for nucleic acid hybridization

Question 14

hybridization with heterologous DNA or with a heterologous probe

Answer 14

• often DNA-DNA hybrids are made when a strand of DNA from one source hybridizes with DNA strand from another source
• such hybrids often have mismatches - places where sequences are not perfectly complimentary

Question 15

Dealing with mismatches

Answer 15

1. make the mismatched hybrids more stable so that you could detect them.
2. prevent hybridization of mismatched hybrids

Question 16

1. make the mismatched hybrids more stable so that you could detect them.

Answer 16

• use gene from one organism to find same gene in DNA of another
• there would be mutations so the genes would not be identical but you could still find them if you did hybridization under conditions that tolerated a few mutation

Question 17

2. prevent hybridization of mismatched hybrids

Answer 17

• make less stable so you would not detect hybrid DNA molecules with mismatches
• find a specific gene among a group of similar genes in an organism to prevent hybridization of DNA except those that match your probe - do this by manipulating.

Question 18

Manipulate the stringency

Answer 18

1. salt concentration

2. temperature

Question 19

Raising salt concentration

Answer 19

• makes the hybrids more stable which lowers stringency

- allows formation of imperfectly matched hybrids

Question 20

Lowering salt concentration

Answer 20

• raises stringency
• make hybrids less stable
• mismatches less well tolerated.

Question 21

lowering the temperature

Answer 21

• makes the hybrids more stable which lowers the stringency

Question 22

raising the temperature

Answer 22

• raise stringency.
• hybrids with mismatches melt more easily
• are less stable.

Question 23

When trying to find the gene from a homologous organism

Answer 23

lower the stringency

Question 24

When trying to find a specific member of a gene family

Answer 24

raise the stringency

Question 25

blot name

Answer 25

• named for the molecule blotted, not the probe

Question 26

Southern blot

Answer 26

• the molecule blotted is DNA
• The probe can be DNA, RNA, or anything else that recognizes DNA
• named for inventor Ed Southern

Question 27

Northern blot

Answer 27

• molecule blotted is RNA

- The probe can be DNA, RNA or anything else that recognizes RNA

Question 28

Western blot

Answer 28

• proteins are blotted
• the probe can be anything that will recognize a specific protein
• generally antibodies.

Question 29

when looking for specific mRNA

Answer 29

• blot and use high stringency conditions
• not a tight and well-defined band because of poly-A tails of varying length so bands are broad and smeary
• mRNA from different family members can have different sizes.

Question 30

Hybridization conditions

Answer 30

• Tm-15 (higher stringency)
• Tm-25 (lower stringency)
• stringency as how many degrees below Tm the hybridization is carried out.

Question 31

common practice

Answer 31

• add a denaturing element to hybridization mixture

- fomamide common - makes hybrid less stable, thus increases stringency