Chapter 21 - Recombinant DNA Technology

Question 1

Define recombinant DNA technology

Answer 1

A general term covering processes by which genes are manipulated, altered or transferred from organism to organisms

Question 2

Define genetically modified organisms

Answer 2

An organisms that has had its DNA altered as a result of recombinant DNA technology

Question 3

Give and briefly describe the five stages of recombinant DNA technology

Answer 3

• Isolation: Production of DNA fragments with the gene
• Insertion: Into a vector
• Transformation: Introduction into host cell
• Identification: Of host cells that have the DNA using gene markers
• Growth/cloning: Bt large scale cultures

Question 4

What are the processess in which we can produce isolated DNA fragments?

Answer 4

• Conversion of mRNA to cDNA using reverse transcriptase
• Using restriction endonuclease to cut fragments with the gene
• Creating a gene with a gene machine

Question 5

How can we use reverse transcriptase to produce genes for RDNAT?

Answer 5

• Cell that produces the protein is selected
• These cells have large quantities of mRNA needed
• Reverse transcriptase used to make cDNA single strand from RNA
• DNA polymerase builds up complementary nucleotides using cDNA as template to produce DNA with target gene

Question 6

Define complementary DNA

Answer 6

DNA that is made from messenger RNA in a process that is the reverse of normal transcription

Question 7

What does reverse transcriptase do?

Answer 7

Catalyse the production of DNA from RNA

Question 8

How do restriction endonucleases produce genes for RDNAT?

Answer 8

Enzymes cut palindromic portions of DNA to leave sticky ends. The gap created is where the gene can be inserted

Question 9

Define palindromic sequence

Answer 9

A section of DNA which reads the same whether read from 5’ to 3’ or 3’ to 5’ direction

Question 10

How does the gene machine manufacture genes?

Answer 10

• Sequence of nucleotides found from protein->mRNA->DNA
• Sequence fed to computer
• Checks for safety/ethics
• Oligonucleotides assembled in sequence
• Gene replicated using PCR which forms DNA

Question 11

Why is the gene machine beneficial to prokaryotes?

Answer 11

No introns are produced

Question 12

How are genes cloned “in-vivo?”

Answer 12

• Isolated DNA fragments and plasmid cut with same restriction enzyme
• DNA inserted and complementary to sticky ends
• Fragments incubated with plasmids and DNA ligase which forms phosphodiester bonds
• RDNA molecule made

Question 13

How are vectors stimulated to take up RDNAT plasmids?

Answer 13

Electroporation

Question 14

How does electroporation work?

Answer 14

• Use of calcium ions and temperature change of 0-40 degrees celsius
• Increases permeability of membranes

Question 15

Why don’t all cells take up RDNAT plasmids in electroporation?

Answer 15

• Only some take up the plasmids when mixed together
• Some plasmids will close up again without the DNA fragment
• DNA fragments form their pwn plasmid

Question 16

What are marker genes?

Answer 16

A separate gene used to identify whether a gene has been taken up bay bacterial cells

Question 17

Give three reasons as to why marker genes are identifiable

Answer 17

• Antibotic resistance
• Fluorescent
• Produces enzyme who’s action is identifiable

Question 18

Summarise the PCR

Answer 18

• DNA sample, primers, DNA polymerase and nucleotides mixed
• Heated to 95 to break hydrogen bonds
• Cooled to 55 so primers can bind
• Heated to 70 for Taq polymerase
• Polymerase creates base pairing using nucleotides
• Repeated around 30 times for sufficient DNA

Question 19

What are primers?

Answer 19

Short sequences of nucleotides that have set bases complementary to those of the two DNA fragments

Question 20

What are the advantages of in vitro cloning?

Answer 20

• Rapid

- No living cells required

Question 21

What are the advantages of in vivo cloning?

Answer 21

• Useful where we wish to introduce genes into organisms
• No contamination
• Accurate
• Cuts specific genes
• Produces bacteria that produce products

Question 22

What is a DNA probe?

Answer 22

Short single stranded length of DNA that has a label, making it identifiable

Question 23

Describe two types of DNA probes

Answer 23

• Radioactive nucleotides with a 32P isotope

- Fluorescent probes which emit light under certain conditions

Question 24

How are DNA probe used to identify alleles?

Answer 24

• DNA probe created complementary to DNA of the allele
• DNA separated
• DNA mixed with probe during DNA hybridisation
• Site at the probe binding is identified by the label on the probe

Question 25

What is DNA hybridisation?

Answer 25

When a section of DNA or RNA is combined with a single strand of DNA which had complementary bases

Question 26

Why is DNA heated in DNA hybridisation?

Answer 26

To separate the two strands of DNA

Question 27

What are VNTRs?

Answer 27

DNA base sequences that do not code and are different in length and number in all individuals

Question 28

State the five stages of genetic fingerprinting

Answer 28

Extraction
Digestion
Separation
Hybridisation
Development

Question 29

How does gel electrophoresis work?

Answer 29

• DNA fragments placed in agar gel
• Voltage applied
• Large moves slow, small moves quickly through the gel
• DNA probes analysed to determine position

Question 30

Summarise the whole process of genetic fingerprinting

Answer 30

• DNA extracted
• DNA cut into blunt end fragments using restriction endonucelases
• DNA separated using gel electrophoresis and in alkali to create single strands. Southern blotting and fixed on to nylon with UV light
• DNA probes added which bind to VNTRs
• X-Ray film placed on nylon membrane to reveal patterns and location of DNA fragments

Question 31

What is southern blotting?

Answer 31

In genetic fingerprinting where the pattern of DNA fragments are transferred to a nylon membrane

Question 32

What are some uses of genetic fingerprinting?

Answer 32

• Crime solving
• Genetic relationships
• Medical Diagnosis
• Plant + Animal Breeding