D1.1 DNA replication

Question 1

Who discovered the DNA structure?

Answer 1

Watson & Crick

Question 2

What did Watson & Crick’s discovery provide evidence for?

Answer 2

Complementary base pairing was the key to DNA’s ability to replicate

Question 3

What is DNA replication?

Answer 3

The process by which exact copies of DNA are created for use in reproduction, growth and tissue replacement in multicellular organisms

Question 4

Why does DNA need to replicate?

Answer 4

1. Cell division - new cells need new DNA for growth & tissue repair
2. Reproduction - gametes requires DNA to pass on genetic info

Question 5

What is the correct theory for DNA replication?

Answer 5

Semi conservative replication

Question 6

How does semi conservative replication work?

Answer 6

1. Hydrogen bonds between the bases are broken
2. Free nucleotides are present in the nucleus
3. Free nucleotides pair up with complementary exposed bases
4. New strand linked together
5. There are now 2 DNA molecules (Each contains one old and one new)

Question 7

What is the function of DNA helicase?

Answer 7

Breaks hydrogen bonds & unwinds molecule (Unzip)

Question 8

What is the function of DNA polymerase?

Answer 8

Form the phosphodiester bonds between nucleotides

Question 9

What are the 3 different theories for DNA replication?

Answer 9

Dispersive
Conservative
Semi conservative

Question 10

What is the evidence for semi conservative replication?

Answer 10

Cultured e coil bacteria in presence of a heavy nitrogen isotope
1. DNA contains nitrogens in its nitrogenous base = radioactive nitrogen will end up in DNA of the bacteria
2. Result: all bacterial DNA had nitrogen in its bases
3. Bacteria all transferred into a fresh medium (nitrogen replaced by a lighter isotope, bacteria was allowed to grow for several generations

Question 11

What is gel electrophoresis used for?

Answer 11

Identify alleles at a few (or few dozen) loci

Question 12

How does gel electrophoresis work?

Answer 12

Uses an electrical current to move molecules through a semi-solid medium
(molecules are usually DNA, RNA or protein - separated by size & amount of charge)

Question 13

What are the key principles of gel electrophoresis?

Answer 13

• DNA & RNA molecules have negative electrical charge = will move towards positive electrode in an electric field
• to get fragments of appropriate size, DNA is digested with restriction endonucleases
• Samples of fragements of DNA -> loaded into wells on one end of the gel
• gel is submerged in buffer solution & electric current is run through the gel
• MUST begin at the negative pole

Question 14

Smaller pieces of fragments in gel electrophoresis travel….

Answer 14

Further along the gel (in a given amount of time), quicker

Question 15

What is used to make DNA have colour? and why?

Answer 15

Ethidium bromide
Binds to DNA, fluoresces in UV light

Question 16

What is a limitation of using ethidium bromide to make DNA have colour?

Answer 16

Mutagenic - safer alternatives used

Question 17

What is the ‘DNA ladder’ for?

Answer 17

It is a DNA size marker, by comparing the position the approximate size can be determined

Question 18

What is PCR and what does it do?

Answer 18

Polymerase Chain Reaction
- a form of artificial DNA replication
- amplifies DNA fragments for further study or processing

Question 19

What does PCR require?

Answer 19

• small DNA sample
• DNA polymerase
• Primers
• Nucleotides
• Thermocycler

Question 20

What is a thermocycler?

Answer 20

Controls and changes its temperature at programmed timings to trigger the different steps in PCR

Question 21

What are the stages in PCR?

Answer 21

Stage 1: Denature to separate DNA strands (95 degrees)
Stage 2: Annealing (joining) of primers to their complementary bases - provides a starting point for DNA polymerase
Stage 3: Synthesis of DNA (temp 72 degrees- optimum temp for DNA polymerase)

Question 22

PCR is a …………….. reaction

Answer 22

Cyclic

Question 23

What are 2 advantages of PCR?

Answer 23

1. Fast
2. Does not require living cells

Question 24

What are some applications of PCR?

Answer 24

• detecting of oncogenes
• detecting mutations
• identify viral infections
• monitoring the effect of a viral disease
• tissue typing
• research
• forensic science

Question 25

What is a DNA profile (DNA fingerprint)?

Answer 25

A unique pattern of genetic markers used to identify an individual

Question 26

How is a DNA profile produced?

Answer 26

1. Extract DNA from sample 2. Digest the sample (cut DNA into small fragments with restriction endonuclease 3. Separate fragments using electrophoresis 4. DNA is separated into single strands (alkaline solution) 5. DNA fragments transferred to nylon membrane 6. Hybridisation DNA probes added (to label) 7. Development - placed onto x ray and developed

Question 27

Uses of DNA profiling

Answer 27

1. Forensic science 2. Maternity/paternity testing 3. Analysis of disease