DNA complementarity hybridisation & its application

Question 1

What is DNA?

Answer 1

Polymer or polynucleotide comprising of 4 nucleotides joined together

Question 2

What does the nitrogenous base comprise of?

Answer 2

A single or double ring, which contains nitrogen and carbon

Question 3

What is attached to carbon 5 of the ribose sugar?

Answer 3

A phosphate group

Question 4

Where is the hydroxy group on the ribose sugar?

Answer 4

Carbon 3

Question 5

Where is the nitrogenous base on the ribose sugar

Answer 5

carbon 1

Question 6

What are the 4 nucleotides?

Answer 6

Cytosine, Guanine, Thymine, Adenine

Question 7

What are the 2 molecular structures that the 4 nucleotides fall into?

Answer 7

Pyrimidines

Purines

Question 8

What are the structural differences between pyrimidines and purines?

Answer 8

Pyrimidine - a single nitrogen ring

Purine- a double nitrogen ring

Question 9

What are the nucleotides that are pyrimidines?

Answer 9

Cytosine and thymine

Question 10

What are the nucleotides that are purines?

Answer 10

Guanine and adenine

Question 11

What are the watson and crick base pairs?

Answer 11

Adenine ad Thymine

Cytosine and Guanine

Question 12

How many hydrogen bonds form between the base pairs?

Answer 12

Guanine and cytosine form 3 hydrogen bonds

Thymine and Adenine form 2 hydrogen base pairs

Question 13

Which base pair is stronger?

Answer 13

Guanine and cytosine because they have 3 hydrogen bonds

Question 14

In RNA which base pair is stronger?

Answer 14

Guanine and Cytosine is still the stronger base pair.

Adenine and Uracil only have 2 base pairs

Question 15

What molecular structure is uracil?

Answer 15

A pyrimidine like Thymine

Question 16

What links the sugar phosphate groups?

Answer 16

Phosphodiester bonds

Question 17

What bonds are present within DNA?

Answer 17

Phosphodiester
For base stacking - Hydrophobic interactions
Van der Waals forces (which are weak but help with stability)

Question 18

What is the form of DNA that is most common?

Answer 18

B form

Question 19

What determines the stability of DNA?

Answer 19

Free energy of the molecule and energy minimisation

Question 20

What can influence the stability of DNA

Answer 20

The molecular environment

Question 21

How is double stranded DNA formed?

Answer 21

From 2 anti parallel strands that means they have opposite orientation

Question 22

What overall charge does DNA have?

Answer 22

Negative due to the phosphate groups. This fact is used for gel electrophoresis

Question 23

DNA denatured

Answer 23

Double strand molecules to a single strand molecule

Question 24

What can cause DNA to denature?

Answer 24

The disruption of Hydrogen bonds within the double helix

Question 25

What causes the hydrogen bonds within DNA to break?

Answer 25

When DNA is heated | Or when DNA is in a strong alkali, formamide or urea

Question 26

What is hyperchromicity?

Answer 26

Increased absorption of light at 260 nm on denaturation

Question 27

What is Tm/ melting temperature?

Answer 27

Point at which 50% of all strands separate.

Question 28

What is special about single stranded DNA?

Answer 28

Hyperchromicity - Single strand DNA absorbs more UV light than double stranded DNA

Question 29

What does Tm depend on?

Answer 29

``` GC content Length of DNA molecule Slat conc (molecular environment) pH (alkali denatures DNA) Mismatched Base pairs ```

Question 30

What does a higher GC content mean?

Answer 30

More Hydrogen bonds = higher Tm

Question 31

What does more hydrogen bonds mean for stability?

Answer 31

There is greater stability?

Question 32

Is there a difference in stability after 300 bp?

Answer 32

No, after 300 bp there is no increase in Tm / stability

Question 33

What cation influences stability?

Answer 33

All of them but sodium ions is the most common

Question 34

What does a higher [Na+] mean for Tm?

Answer 34

A higher Tm, as salt conc increases stabilisation. | Higher Salt conc also over comes how unstable mismatched base pair are

Question 35

What else does a high salt also do?

Answer 35

Reduces the specificity of base pairing

Question 36

What does an alkali solution do to DNA?

Answer 36

Alkali dissociates into its ions. | The OH- ion disrupts the hydrogen bonds in DNA

Question 37

Higher pH means?

Answer 37

A lower Tm

Question 38

What is mismatch?

Answer 38

A base pair combination that is unable to form a hydrogen bond

Question 39

Low no of H bonds?

Answer 39

Low Tm

Question 40

What else does DNA mismatch do to DNA?

Answer 40

Distorts the structure and destabilise adjacent base pairing

Question 41

How do you reverse denaturation?

Answer 41

Slow cooling | Neutralisation

Question 42

What is renaturation / hybridisation?

Answer 42

Formation of duplex structure of two DNA molecules that have been introduced to one another

Question 43

Are there different factors that influence renaturation and hybridisation?

Answer 43

No they are both examples of duplex formation

Question 44

What contributes to a high Tm?

Answer 44

Perfect base pairs

Question 45

What is stringency?

Answer 45

Manipulating conditions: Limiting hybridisation between imperfectly matched sequences allows us to manipulate specificity

Question 46

Why would we want low stringency?

Answer 46

Kinetics of hybridisation are much faster under low stringency conditions

Question 47

What determines high stringency?

Answer 47

Temperature near the Tm or a low salt conc

Question 48

What are the 6 nucleic acid based techniques?

Answer 48

- Northen blotting - Southern blotting - Microarrays - PCR - Cloning - Dideoxy and next gen sequencing

Question 49

Specific aspects of Hybridisation techniques?

Answer 49

Identifies the presence of NA containing a specific sequence of bases

Question 50

What are uses of probes?

Answer 50

Capture specific species of DNA Amplify segments of DNA Or to quantify the no of molecules present

Question 51

What is a probe?

Answer 51

ssDNA or RNA About 20 -1000 bases in length Labelled with a fluorescent or luminescent molecule

Question 52

The limitation of northern blotting

Answer 52

One gene at a time and a small sample size messy and time consuming PCR is better

Question 53

Northern and southern blotting

Answer 53

- Uses DNA or RNA that has been separated by gel electrophoresis. - Transferred to a mass capillary flow of a buffer from a reservoir with nylon membrane - Captured by and covalently bonded to the membrane and then hybridised with a labelled probe

Question 54

Microarrays

Answer 54

An ordered assembly of thousands nucleic acid probes | -They are fixed to a solid surface, then the sample of interest is hybridised to the probes

Question 55

Where are the probes fixed to?

Answer 55

A solid surface like a glass matrix or silicon

Question 56

What are microarrays used for?

Answer 56

Gene expression profiling for drug treated cells and untreated cells

Question 57

What are GWAS?

Answer 57

Genome Wide Association studies | Microarrays with SNPs are used for GWAS

Question 58

What is needed for microarrays?

Answer 58

RNA Labelled with a fluorescent molecule hybridised to the array and amount and location of the label measured