DNA manipulation

Question 1

Define restriction enzymes

Answer 1

Are groups of enzymes that cut a strand of DNA into one or more peices. They do this by breaking the phosphodiester linkage between different DNA nucleotides

Question 2

Restriction enzymes cut at…

Answer 2

the region of a specific base sequence (recognition site) . Note that the base sequence is palindromic (sequence of one strand needs to be the same as the complementary base sequence read backwards)

Question 3

Restriction enzymes

Define blunt ends

Answer 3

Strands of DNA are cut at the same point, so there are no single-stranded DNA with a complementary base sequence.

Question 4

Restriction enzymes

Define sticky ends

Answer 4

The two strands of DNA is cut at different points. meaning that each piece has a single stranded end which they could easily stick into and anneal with another single-stranded end with a complementary base sequence

Question 5

Function of ligase

Answer 5

Ligase is an enzyme that can stick two single strands of DNA together to form one longer strand. It does this by catalysing the formation of phosphodiester linkages between DNA strands.

Question 6

PCR (polymerase chain reaction)

Purpose

Answer 6

Is a process used to amplify a small sample of DNA. It involves cycling the DNA through 3 different temperatures in the presence of Taq Polymerase (extremely high optimal temp) , DNA primers and nucleotides(A, T, C,G)

Question 7

PCR (polymerase chain reaction)

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Answer 7

• Heat the DNA sample to 94°C for two minutes. This will cause the DNA to denature
• Cool the mixture down to 55°C for two minutes. This will allow the short DNA primers to anneal to complementary segment of the single- stranded DNA.
• Heat the mixture to 72°C for one minute. This will allow the Taq polymerase to synthesise a new strand of DNA from the primer, using the original strands of DNA as a template.

Question 8

Pop quiz

PCR (polymerase chain reaction)
After 5 cycles how many pieces of DNA will there be

Answer 8

32

Question 9

Gel electrophoresis

Purpose

Answer 9

Sort DNA fragments according to their length

Question 10

Gel electrophoresis

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Answer 10

• The voltage of the power supply
• The density of the agarose gel
• The size (or molecular weight) of the DNA fragments- The gel is porous so dna can move through, also dna has a negative charge due to phosphate group therefore it tends to move to the positive terminal on other side of gel

Question 11

Define vector

Answer 11

is anything that can be used by a genetic engineer to transport ‘passenger’ DNA into a cell. This can be a virus, plasmid or liposome.

Question 12

Use of vectors

Define plasmid

Answer 12

Are small circular loops of DNA. They are found naturally in bacteria. They have a few genes on them.

Question 13

Use of vectors

Function of bacteria relating to plasmid

Answer 13

Bacteria can be made to take a plasmid up, by soaking the bacteria in a salty solution and heat shocking them.

Question 14

Define recombinant dna technology (genetic engineering)

Answer 14

Involves cutting a gene out of one species using restriction enzymes, and incorporating it in the genome of another species so that the other species express whatever characteristics the gene codes for.

Question 15

Recombinant dna technology (genetic engineering)

How is it possible

Answer 15

Because the genetic code is universal (same in all species)

Question 16

Recombinant dna technology (genetic engineering)

Why does it sometimes not work?

Answer 16

Because the functional protein produced depends on not only order of amino acids but also the way the protein is modified by other enzyme. If these enzymes arent present, the tertiary or quartnary structure may be different therefore other modifications wont be made.