Recombinant DNA

Question 1

What is recombinant DNA technology?

Answer 1

The transfer of fragments of DNA from one organism / species, to another.

Question 2

Give two reasons why bacteria are able to use human DNA to produce human proteins (2 marks).

Answer 2

1. The genetic code is universal
2. The mechanism of transcription is universal;
3. The mechanism of translation is universal;

Question 3

List the 5 steps in recombinant DNA technology in the correct order

Answer 3

1. Isolation of DNA (usually contains a gene)
2. Insertion of DNA into a vector (e.g. a plasmid)
3. Transformation of cells (to produce a genetically modfied or transgenic organism with two or more sources of DNA).
4. Identification of cells that have taken up the DNA by using marker genes
5. Growth / cloning
   - i.e. bacterial divide by binary fission
   - amplify DNA using PCR

Question 4

3 ways to isolate a DNA fragment
which typically contains a gene

Answer 4

1. Reverse transcriptase & mRNA
2. Restriction endonucleases
3. Gene machine

Question 5

Enzyme required to convert
mRNA into cDNA

Answer 5

reverse transcriptase

Question 6

Describe how isolated mRNA from a cell can be converted into DNA (3 marks).

Answer 6

1. mRNA is mixed with free DNA nucleotides AND reverse transcriptase.
2. Free DNA nucleotides bind to single stranded mRNA template via complementary base pairing.
3. Reverse transcriptase joins DNA nucleotides together to form a single stranded cDNA molecule.
4. DNA polymerase is required to make cDNA double stranded.

cDNA means copy DNA i.e. it is a copy based on mRNA

Question 7

Advantages of using reverse transcriptase to isolate DNA fragments

Answer 7

Introns have been removed
Cells producing protein will contain many mRNA molecules
mRNA is easy to isolate from cells

Question 8

Disadvantages of using reverse transcriptase to isolate DNA fragments

Answer 8

Many steps involving involving enzyme-controlled reactions
Time consuming
Requires more technical expertise

Question 9

name of enzyme that isolate a gene / section of DNA from a larger section of DNA e.g. a chromosome.

Answer 9

Restriction endonucleases

Question 10

Restriction endonucleases are enzymes that [1] phosphodiester bonds at specific DNA base sequences called [2] sites.

Answer 10

hydrolyse
restriction

Question 11

Restriction sites are often _______________

Answer 11

palindromic

Question 12

What is the consequence if the restriction site for the restriction endonuclease occurs within the DNA fragment researchers wish to isolate.

Answer 12

This will cut the gene and it will not code for a functional protein.

Question 13

What type of ends are produced after cutting DNA with restriction endonucleases.

Answer 13

Sticky ends

Blunt ends

Question 14

What are sticky ends used for?

Answer 14

To insert a gene into a vector
(e.g. a plasmid)

Question 15

What are blunt ends used for?

Answer 15

Can be amplified by the polymerase chain reaction;

Separated by size using gel electrophoresis;

Question 16

Advantages of using restriction endonucleases to isolate DNA fragments

Answer 16

• Produce sticky and blunt ends
• 1000s of restriction endonucleases have been isolated that are each highly specific to different DNA sequences

Question 17

Disadvantages of using restriction endonucleases to isolate DNA fragments

Answer 17

contains introns

Enzymes may cut in the middle of the desired gene leading to a non-functional protein

Question 18

Which method of DNA isolation is decribed below:

Desired sequence of nucleotide bases are entered into a computer and automated machinery synthesises the DNA fragment

Answer 18

Gene machine

Question 19

Advantages of using gene machines to isolate DNA fragments

Answer 19

• Faster process owing to automated machinery and fewer enzyme controlled reactions
• Sequences contain no introns
• Blunt and sticky ends can be added

Question 20

Disdvantages of using gene machines to isolate DNA fragments

Answer 20

If sequence of DNA is unknown, requires the primary structure of the polypeptide to be known.

Question 21

Once a DNA fragment has been isolated, what must be added to enable gene expression?

Answer 21

A promoter region
(allow transcription factors to bind)
A terminator region
(ensures only the DNA fragment is transcribed)

Question 22

Suggest and explain one reason why bacteria might not be able to produce every human protein (1 mark).

Answer 22

Cannot splice pre-mRNA, so cannot remove introns

OR

Do not have Golgi apparatus, so cannot process/modify proteins;

OR

Do not have the required transcriptional factors, so cannot carry out transcription/produce mRNA;

Question 23

A geneticist concluded it would be faster to create a human gene using a gene machine than by using reverse transcriptase to convert mRNA into cDNA.

Suggest why the geneticist reached this conclusion.

Answer 23

faster to use gene machine than all the enzyme-catalysed reactions (involving reverse transcriptase);

Question 24

Which methods of DNA isolation could used prior to insertion of the human gene into a bacteria to ensure the protein could be produced.

Answer 24

1. mRNA & reverse transcriptase AND gene machine
2. Produce DNA / human gene without introns;
3. Bacteria cannot remove introns / cannot splice mRNA / cannot splice pre-mRNA;

Question 25

1. mRNA & reverse transcriptase AND gene machine 2. Produce DNA / human gene without introns; 3. Bacteria cannot remove introns / cannot splice mRNA / cannot splice pre-mRNA;

Answer 25

A DNA carrier that can be used to transfer foreign DNA into cells

Question 26

Examples of vectors

Answer 26

Bacterial plasmid; Viruses

Question 27

For insertion to occur, the DNA fragment and plasmid must be cut with the ___________ restriction endonuclease.

Answer 27

same

Question 28

Describe how enzymes could be used to insert a human gene into a plasmid (2 marks).

Answer 28

1. Cut the plasmids with the same restriction endonuclease (used to isolate the gene); 2. Both have complementary sticky ends; 3. (Mix together) and add DNA ligase to join human gene to the plasmid vector (via phosphodiester bonds);

Question 29

What bond forms between the sticky ends?

Answer 29

Hydrogen bonds via complementary base pairing

Question 30

What bond does DNA ligase form to join the isolated DNA to the plasmid?

Answer 30

Phosphodiester

Question 31

Describe how a bacterial cell be transformed?

Answer 31

Heat or electric shock; Creates short-lived pores in the cell surface membrane; Allows plasmids (with DNA of interest) to be taken up;

Question 32

Describe binary fission in bacteria (3 marks).

Answer 32

1. Replication of circular DNA; 2. Replication of plasmids; 3. Division of cytoplasm (to produce daughter cells);

Question 33

Why are not all cells transformed?

Answer 33

1) Not all the vectors take up DNA to become recombinant (unsuccessful insertion) 2) Not all the cells take up recombinant vectors (unsuccessful transformation).

Question 34

What type of gene is need to identify if a cell has successfully been transformed / is transgenic / genetically modified?

Answer 34

Marker gene

Question 35

Define a marker gene

Answer 35

allow easy identification of cells that have taken up the vector (e.g. plasmid) with the DNA fragment / gene of interest

Question 36

3 examples of marker genes

Answer 36

1. Green flourescent protein (GFP) 2. Antibiotic resistance 3. Lactase (which produces a blue product)

Question 37

What happens if a gene of interest is inserted into the middle of the ampicillin resistance gene in a plasmid?

Answer 37

Damages the gene; Codes for non-functional protein; Bacteria will be destroyed if explosed to amplicillin (no longer resistant to antibiotic);

Question 38

To enable researchers to identify which bacteria have taken up the plasmid with a human gene, the plasmids contain a gene that codes for a green fluorescent protein (GFP). Bacteria that contain this plasmid glow green under UV light. Suggest one advantage of using this gene for GFP to identify bacteria that have taken up plasmids (1 mark).

Answer 38

Can quickly identify transformed bacteria using UV light;

Question 39

Define in vivo cloning and provide examples

Answer 39

Using a living organism to produce genetically identifcal daugher cells e.g., bacteria, goat, fish, crop plant

Question 40

What does PCR stand for?

Answer 40

polymerase chain reaction

Question 41

is PCR an example of in vitro or in vivo cloning of DNA

Answer 41

in vitro

Question 42

The polymerase chain reaction is used to ____________ . a

Answer 42

amplify DNA

Question 43

What temperature is required to break the hydrogen bonds in DNA during PCR?

Answer 43

95oC

Question 44

What is a primer?

Answer 44

Short single stranded DNA molecule; Has complementary bases to the start of the DNA fragment; Extended by thermostable DNA polymerase;

Question 45

What temperature is required for the ‘annealing’* of primers to single stranded DNA? *annealing is primers binding to each end of the template strands

Answer 45

55oC

Question 46

What type of DNA polymerase is required for PCR?

Answer 46

thermostable

Question 47

What temperature is used during the synthesis stage of PCR?

Answer 47

72oC

Question 48

TRUE or FALSE: Thermostable DNA polymerases denatures at 72oC?

Answer 48

FALSE

Question 49

Describe how an isolated gene can be replicated by the polymerase chain reaction (PCR) (4 marks).

Answer 49

1. Heat DNA to 95oC to break (weak) hydrogen bonds / separate strands 2. Add primers and add DNA nucleotides 3. Cool to 55oC to allow ‘annealing’ of primers and DNA nucleotides via complementary base pairing 4. Add thermostable DNA polymerase (e.g. Taq polymerase); 5. Heat to 72oC 6. DNA polymerase joins nucleotides together by phosphodiester bonds to synthesise new strand by extending the primers;

Question 50

Which formula can be used to calculate the number of DNA strands produced after PCR?

Answer 50

2n

Question 51

Assuming we started with one DNA molecule, how many DNA molecules are produced after 25 PCR cycles?

Answer 51

2^25 = 3.3x10^7

Question 52

Assuming we started with 15 DNA molecules, how many DNA molecules are produced after 25 PCR cycles?

Answer 52

15 x 2^25 = 5 x 10^8

Question 53

How many PCR cycles produced 7230124899 DNA molecules?

Answer 53

log2(7230124899) = 32.8

Question 54

Why does the number of DNA molecules produced eventually plateau after many PCR cycles?

Answer 54

DNA nucleotides being used up; so less / nothing to make complementary strands; OR Primers used up; so thermostable DNA POLYMERASE cannot start complementary strands;

Question 55

Benefits of recombinant DNA technology

Answer 55

Replace faulty or lack of protein (e.g. insulin in type 1 diabetes) Gene therapy (e.g. correct for mutation or lack of gene expression in an organsim) Improve agriculture (e.g. larger yields, disease resistance, drought tolerance) Improve industrial processes (e.g. produce larger quantities of enzymes more quickly OR improve enzyme-controlled rate of reaction) Improve understanding of biological processes (e.g. the regulation of gene expression, DNA replication, mitosis and cell death)

Question 56

Concerns regarding the widespread use of recombinant DNA technology

Answer 56

1. Inserting new genes into a crop plant could disrupt other gene/s function creating toxic products within genetically modified food sources. 2. Introducing herbicide resistance genes to crop plants could result in transfer to wild species when they interbreed. 3. Technology may become concentrated in the hands of large corporations. (e.g. technology improves health outcomes but is not accessible to socially and economically disadvantaged groups).

Question 57

What is gene therapy?

Answer 57

Introduction of healthy gene (which codes for functioning protein) into defective cells (caused by a mutation)

Question 58

Vectors for gene therapy in humans

Answer 58

Viruses Liposomes

Question 59

Describe how a virus acts as a vector in gene therapy.

Answer 59

1. Insert isolated DNA fragment or gene of interest into viral genome. 2. Virus will insert this gene at the same time as its own genetic material into hosts cells during infection. 3. Host cell transcribes and translates gene that codes for functioning protein during replication

Question 60

Disadvantage of using viral vectors

Answer 60

1. May cause an immune response e.g. formation of cytotoxic T cells / B cells / memory cells 2. Not all host cells are successfully infected with the genetically modified virus

Question 61

Advantage of using viral vectors

Answer 61

1. Can enter cells / infect cells / inject DNA into cells; 2. Targets specific cells (attachment proteins bind to receptors); 3. Replicates in cells

Question 62

Describe how a liposome acts as a vector in gene therapy.

Answer 62

1. Lipid soluble 2. So cross the phospholipid bilayer (and release DNA / gene into cells)

Question 63

Suggest why the plasmids which contain a gene of interest from a spider - that codes for protein that produce stronger silk fibres - were injected into the eggs of silkworms, rather than into the cells of adult silkworms (2 marks).

Answer 63

1. Gene gets into all / most of cells of silkworm; 2. So gets into cells that make silk.

Question 64

The scientists ensured the spider gene - that codes for stronger silk fibre proteins - was expressed only in cells within the silk glands of the silkworm. What would the scientists have inserted into the plasmid along with the spider gene to ensure that the spider gene was only expressed in the silk glands of the silkworms?

Answer 64

Promoter

Question 65

the scientists ensured the spider gene - that codes for stronger silk fibre protiens - was expressed only in cells within the silk glands of the silkworm. Suggest two reasons why it was important that the spider gene was expressed only in the silk glands of the silkworms.

Answer 65

1. So that silk fibre protein can be harvested; 2. Silk fibre proteins in other cells might cause harm;