DNA Hybridization

Question 1

What is a pentose sugar?

Answer 1

→ 5 carbons that form a cyclical structure with an oxygen bridge

Question 2

What are the pyrimidines?

Answer 2

→ Cytosine
→ Thymine
→ Uracil

Question 3

What are the purines?

Answer 3

→ Adenine

→ Guanine

Question 4

On what basis does hydrogen bonding form?

Answer 4

→ Watson and Crick base pairing

Question 5

Between what two groups does bonding occur?

Answer 5

→ Purines and pyrimidines

Question 6

How many hydrogen bonds are there between T and A?

Answer 6

→ 2

Question 7

How many hydrogen bonds are there between C and G?

Answer 7

→ 3

Question 8

Between what base pairs is hydrogen bonding the strongest and why?

Answer 8

→ C and G

→ 3 H bonds

Question 9

What are sugar phosphates linked by?

Answer 9

→ phosphodiester bonds

Question 10

What kind of interactions does base stacking involve?

Answer 10

→ hydrophobic interactions

Question 11

Why are bases stacked?

Answer 11

→ To exclude water from the inside of the molecule around the bases

Question 12

Describe what the Van der Waals forces are like in DNA?

Answer 12

→ Individually small but they contribute to the stability

Question 13

What is the backbone of DNA formed by?

Answer 13

→ Phosphodiester linkage

Question 14

What do phosphodiester bonds join?

Answer 14

→ 3 and 5 prime carbons of deoxyribose of DNA

Question 15

What does the stability of DNA depend on?

Answer 15

→ Free energy of the molecule

→ Energy minimization

Question 16

What do phosphate groups interact with?

Answer 16

→ proteins

Question 17

What bonds can be broken to denature DNA?

Answer 17

→ Hydrogen bonds

Question 18

What disrupts hydrogen bonding?

Answer 18

→ Alkalis

→ Heat

Question 19

What is hyperchromicity?

Answer 19

→ Increased absorption of light at 260nm on denaturation

Question 20

What is Tm?

Answer 20

→ The point at which 50% of all strands separate is called the melting temperature

Question 21

What increases as the molecule is denatured?

Answer 21

→ Absorption

Question 22

What 5 things affect Tm?

Answer 22

→ GC content
→ Length of molecule
→ Salt concentration
→ pH
→ Mismatches

Question 23

What does a higher Gc content mean?

Answer 23

→ more hydrogen bonds

→ Higher Tm

Question 24

What is the relationship between the length of DNA and Tm?

Answer 24

→ The longer the contiguous duplex the higher the Tm

Question 25

Up until what point does adding more hydrogen bonds increase stability?

Answer 25

→ up until 300 bp

Question 26

What does high salt do to DNA duplexes?

Answer 26

→ stabilizes them

Question 27

What is the relationship between Na+ and Tm?

Answer 27

→ High Na+ = High Tm

Question 28

What does increasing the salt concentration do to the stability and Tm?

Answer 28

→ Stabilizes the structure and increases the Tm | → overcomes the effect of mismatched base pairing b

Question 29

What does increasing salt do to base pairing?

Answer 29

→ Reduces the specificity of base pairing at a given temperature

Question 30

What are 3 chemicals that disrupt hydrogen bonds?

Answer 30

→ urea → formamide → alkali

Question 31

What kind of pH destabilizes DNA duplexes?

Answer 31

→ high

Question 32

What is a mismatch?

Answer 32

→ A base pair that is unable to form hydrogen bonds

Question 33

Why do mismatches weaken DNA?

Answer 33

→ It reduces the number of hydrogen bonds | → Fewer H bonds means a lower Tm

Question 34

How do mismatches destabilize base pairing?

Answer 34

→ they allow other molecules to penetrate within the structure of the molecule

Question 35

What is the reverse of denaturation called?

Answer 35

→ Renaturation

Question 36

What is renaturation facilitated by?

Answer 36

→ Slow cooling and neutralization

Question 37

What is hybridization?

Answer 37

→ Renaturation but with the introduction of a foreign molecule into a solution

Question 38

Why is renaturation favored?

Answer 38

→ Formation of the structure favors energy minimization which is driven by a change in free energy delta G

Question 39

Why do perfect matches have a higher Tm?

Answer 39

→ They are thermodynamically favored over mismatches

Question 40

How can you manipulate specificity?

Answer 40

→ Limiting hybridization between imperfectly matched sequences allows to manipulate specificity

Question 41

What is high stringency?

Answer 41

→ Temperature near the Tm | → Low salt concentration

Question 42

How does nucleic acid hybridization work?

Answer 42

→ Identifies the presence of nucleic acid containing a specific sequence of bases →A labeled molecule is used to investigate a mixed population of molecules to find out how many of a specific sequence there are

Question 43

How do you find a specific strand within DNA with a probe?

Answer 43

→ Label a probe and hybridize it with a strand you are looking for that corresponds to a gene

Question 44

What is a probe?

Answer 44

→ A single strand of DA | → Labelled with a fluorescent molecule

Question 45

Why is PCR used over gel techniques?

Answer 45

→ Gel techniques are messy

Question 46

How does a microarray work?

Answer 46

→ Ordered assembly of probes are attached to the surface of a glass matrix → Hybridization is used to attach the complementary molecules to the surface of the matrix → This can measure 50,000 transcripts in a cell

Question 47

What are SNPs?

Answer 47

→ Single nucleotide polymorphisms are the most common type of genetic variation among people → Each SNP represent a difference in a single nucleotide