Recombinant DNA Technology

Question 1

what is recombinant DNA technology

Answer 1

• the transfer of fragments of DNA from one organism to another

Question 2

why does recombinant DNA technology work?

Answer 2

• the genetic code is universal
• the transcription mechanism is universal
• the translation mechanism is universal

Question 3

what is a transgenic organism

Answer 3

• an organism that has received transferred DNA

Question 4

what methods are used to obtain the fragments of DNA (genes) used in RDT

Answer 4

• Using Reverse Transcriptase
• Using a Gene Machine
• Using Restriction Endonucleases

Question 5

Describe how reverse transcriptase is used to obtain DNA fragments used in RDT

Answer 5

• mRNA,free DNA nucleotides and reverse transcriptase are mixed together
• the mRNA transcribed from the gene is used as a template
• the free DNA nucleotides line up next to their complementary bases on the mRNA template
• reverse transcriptase then joins the DNA nucleotides together to produce the required fragment of DNA called complementary DNA (cDNA)
• double stranded DNA is produced from this cDNA using DNA nucleotides and DNA polymerase

Question 6

Describe how restrictive endonuclease is used to obtain DNA fragments used in RDT

Answer 6

• restrictive endonucleases hydrolyse the DNA at specific recognition sites cutting out the required DNA fragment.

Question 7

Describe how a gene machine is used to obtain DNA fragments used in RDT

Answer 7

• amino acid sequence is used as a template to determine the sequence of DNA nucleotides for a specific gene

Question 8

what are the advantages of using reverse transcriptase for RDT

Answer 8

• mRNA is present in large amounts in protein-making cells
• Absence of Introns

Question 9

what are the advantages of using a gene machine for RDT

Answer 9

• the process is automated
• faster than other methods as its not enzyme controlled

Question 10

when must introns not be present during RDT

Answer 10

• if the source of a gene being transferred is eukaryotic and the intended recipient is prokaryotic

Question 11

what sections of the DNA must be added to the fragment of DNA during RDT for successful transcription of the transferred genes

Answer 11

• promotor regions which initiate transcription of the gene by promoting the binding of RNA polymerase
• Terminator regions which mark the end of a gene and trigger the release of the mRNA transcribed

Question 12

how can the amplification (increase in number via replication) of the fragments of DNA be achieved?

Answer 12

• in vivo - the copies are made inside a living organism
• in vitro - the copies are made outside of a living organism

Question 13

what are sticky ends and blunt ends?

Answer 13

sticky ends - DNA hydrolysed at different locations resulting in exposed bases
blunt ends - DNA hydrolysed at the same location resulting in no exposed bases

Question 14

where do restriction enzymes hydrolyse RNA/DNA

Answer 14

• at specific recognition sequences/sites

Question 15

describe the process of the Polymerase Chain Reaction

Answer 15

stage 1
- sample DNA, DNA primers, free DNA nucleotides and DNA polymerase are mixed together and heated at 95°C for 5 minutes breaking the hydrogen bonds in DNA

stage 2
- mixture is cooled to 50-60°C allowing the primers to join to their specific complementary target sequence
- free DNA nucleotides align to the DNA strands by complementary base pairing

stage 3
- the temperature is increased to 72°C=optimum for DNA polymerase
- enzyme joins the individual nucleotides of a strand together to form a new complementary strand

Question 16

how to work out number of DNA molecules formed via PCR

Answer 16

2ⁿ , n = number of cycles

Question 17

what are vectors

Answer 17

• how genes are transferred into different cells

Question 18

what are the two types of vectors

Answer 18

• plasmids
• bacteriophages

Question 19

define transformation

Answer 19

• the uptake of recombinant plasmids by a culture of bacteria

Question 20

why are vectors not guaranteed to work

Answer 20

• the cells may not take up the vector at all
• the plasmid may have joined back together without the gene being taken up
• the DNA fragment may have annealed to itself

Question 21

describe how recombinant plasmids are made?

Answer 21

• a plasmid is cut using the same restriction endonucleases used to cut the gene
• the plasmid DNA and foreign DNA join by complementary base pairing due to their complementary sticky ends
• the enzyme ligase is used to form phosphodiester bonds

Question 22

what are marker genes

Answer 22

• genes that enable successfully transformed bacteria or eukaryotic cells to be detected and isolated

Question 23

one example of a marker gene is…

Answer 23

• GFP gene which codes for the production of a green fluorescent protein causing successfully transformed cells to be identified as they fluoresce when viewed with UV light under a microscope

Question 24

what are the pros of the use of Recombinant DNA Technology

Answer 24

• used to reduce famine and malnutrition by developing genetically modified plants or animals which produce high yields and are resistant to disease
• used to produce vaccines and drugs
• used to treat genetic diseases by gene therapy

Question 25

what are the cons of the use of Recombinant DNA Technology

Answer 25

• can lead to possible transfer of foreign genes to non-target organisms
• it is an irreversible process
• there are ethical considerations with regards to permanently altering the genome of animals
• long term ecological and evolutionary consequences are unknown