DNA and hybridisation

Question 1

what do nucleotides make up?

Answer 1

DNA and RNA

Question 2

What does DNA compromise of?

Answer 2

4 nucleotides joined together.

• nitrogenous base
• ribose sugar
• phosphate group

Question 3

what are the 4 nucleotides that DNA consist of?

Answer 3

cytosine
thymine
guanine
adenine

Question 4

which nucleotides are purines

Answer 4

adenine

guanine

Question 5

which nucleotides are pyrimidines

Answer 5

thymine

cytosine

Question 6

how many hydrogen bonds between cytosine and guanine

Answer 6

3

Question 7

how many hydrogen bonds between thymine and adenine

Answer 7

2

Question 8

describe DNA structure

Answer 8

antiparallel strands
negatively charged phosphates on outside
bases on inside

Question 9

at what absorbance can denaturation be optically measured

Answer 9

260nm

Question 10

what is the melting temperature (Tm)

Answer 10

the point at which 50% of all strands seperate

Question 11

what does TM depend on? (6)

Answer 11

hydrogen bonds
GC content
length of DNA molecule
salt conc
PH
mismatches

Question 12

what does more GC mean for Tm

Answer 12

more GC = more H bonds = higher Tm

Question 13

what does longer molecule length mean for Tm

Answer 13

higher tm, due to more h bonds in longer molecule = more strength.

stops past 300bp

Question 14

what does high salt conc mean for Tm

Answer 14

higher tm

high salt conc = stable structure = overcomes destabilising effect of mismatched base pairs

Question 15

what does less hydrogen bonds do to the Tm?

Answer 15

lowers Tm

Question 16

define mismatch

Answer 16

a base pair combination that is unable to form hydrogen bonds

Question 17

do mismatches lower Tm

Answer 17

yes
-reduces no. of h-bonds
shorter stretches of double stranded sequences

Question 18

what is reversal of denaturation?

Answer 18

formation of structure favours energy minimisation driven by change in free energy

Question 19

similarities in renaturation and hybridisation?

Answer 19

-involve DNA molecules that have been introduced to each other
both duplex formation

Question 20

why do complementary bases have higher tm

Answer 20

thermodynamically favoured

no mismatching

Question 21

high stringency on tm?

Answer 21

higher tm

stable complementary base pairing

Question 22

application of complimentarity and hybridisation?

Answer 22

northern blotting
southern blotting
microarrays
dideoxy and next gen sequencing
PCR
cloning

Question 23

what does nucleic acid hybridisation allow?

Answer 23

identifying presence of nucleic acids containing specific sequence of bases
allows absolute or relative quantitation of these sequences in a mixture

Question 24

what is a probe

Answer 24

single stranded DNA or RNA
between 20-1000 bases
labelled with fluorescent molecule

Question 25

nucleic acid blotting techniques

Answer 25

northern blotting

analysis of mana or DNA

Question 26

flaws of nucleic acid blotting techniques

Answer 26

limited technology = detect one gene at a time
small no of samples
gel based techniques are messy and long

Question 27

explain northern/southern blotting

Answer 27

1. extract RNA or DNA
2. gel electrophoresis
3. transfer to nylon membrane
4. add labeled probe, hybridises mRNA
5. detect hybridisation
6. nylon membrane captures nucleic acid
7. mass flow through gel capillary action carries nucleic acid

Question 28

what are microarrays?

Answer 28

ordered assembly of thousands of nucleic acid probes

Question 29

how do microarrays work?

Answer 29

1. probes fixed to solid surface
2. sample is hybridised to probes
3. RNA extracted, labelled and hybridised.

used to compare gene expression

Question 30

microarrays and SNPs

Answer 30

Detects 2.5 million SNPs simultaneously

• used in genome wide association studies