Recombinant DNA technology

Question 1

How were human diseases treated before DNA technology?

Answer 1

A number of diseases result from being unable to produce various metabolic chemicals.
Many of these are proteins, so are the product of a gene.
Treatment previously involved extracting the chemical from a donor and introducing it to the patient, which risked rejection and risk of infection, and is expensive.

Question 2

What is recombinant DNA technology?

Answer 2

There are advantages to produce large quantities of proteins, so techniques have been developed to isolate genes, clone and transfer them into microorganisms.
These are then grown to provide continuous production of the protein.
The DNA from 2 different organisms that has been combined is recombinant DNA.
The resulting organism is transgenic or genetically modified organism.

Question 3

Why is DNA of an organism accepted by a different species and functions normally?

Answer 3

The genetic code is the same in all organisms (universal).
Making proteins is also universal in that the mechanisms of transcription and translation are the same in all organisms.
So transferred DNA can be transcribed and transferred in the cells of the transgenic organism and proteins coded for in the same way.

Question 4

What are the stages of using DNA technology of gene transfer and cloning?

Answer 4

Isolation of the DNA fragments that have the gene for the desired protein.
Insertion of the fragment into a vector.
Transformation - transfer of DNA into suitable host cells.
Identification of the host cells that have successfully taken up the gene by gene markers.
Cloning/growth of the population of host cells.

Question 5

What are the methods of producing DNA fragments?

Answer 5

Conversion of mRNA to cDNA using reverse transcriptase.
Using restriction endonucleases to cut fragments containing the desired gene from DNA.
Creating the gene in a gene machine, based on known protein structure.

Question 6

What are retroviruses?

Answer 6

A group of viruses e.g. HIV, where the coded information is RNA.
In a host cell they are able to synthesise DNA from their RNA using the enzyme reverse transcriptase, which catalyses the production of DNA from RNA.

Question 7

How is reverse transcriptase used to isolate a gene?

Answer 7

A cell that readily produces the protein is selected. (E.g. B cells of islets of langerhans from the pancreas to produce insulin).
These cells have large quantities of the relevant mRNA, which is more easily extracted.
Reverse transcriptase makes complementary DNA from RNA. cDNA is called because it is made up of the nucleotides that are complementary to the mRNA.
To make the other DNA strand, DNA polymerase builds up the complementary nucleotides on the cDNA template.

Question 8

How do bacteria use restriction endonucleases?

Answer 8

Bacteria are frequently infected by viruses that inject their DNA into them in order to take over the cell.
Some bacteria defend themselves by producing enzymes that cut up the viral DNA - restriction endonucleases.

Question 9

What are restriction endonucleases?

Answer 9

Each type cuts a DNA double strand at a specific sequence of bases called the recognition sequence.
Sometimes this cut occurs between two opposite base pairs.
This leaves two straight edges - blunt ends.

Question 10

What do other types of restriction endonucleases do?

Answer 10

They cut DNA staggered, leaving an uneven cut in which each strand of the DNA has exposed, unpaired bases - sticky ends.
The sequences of unpaired bases that remain are opposites of on another - a palindrome.

Question 11

How does the gene machine work (1)?

Answer 11

The desired sequence of nucleotide bases is determined from the desired protein. The amino acid sequence of the protein is determined, and the mRNA codons looked up and cDNA triplets worked out.
The desired sequence of nucleotide bases is fed into a computer and checked for biosafety and biosecurity to ensure it meets international and ethical standards.

Question 12

How does the gene machine work - oligonucleotides (2)?

Answer 12

The computer designs a series of small, overlapping single strands of nucleotides - oligonucleotides, which can be assembled into the desired gene.
In an automated process, each oligonucleotide is assembled by adding one nucleotide at a time in the required sequence.
The oligonucleotides are then joined to make a gene, then replicated using polymerase reaction.
This also constructs the complementary strand of nucleotides to make the double stranded gene, then multiplied to give many copies.

Question 13

How does the gene machine work - insertion (3)?

Answer 13

Using sticky ends, the gene can be inserted into a bacterial plasmid, which acts as a vector for the gene to be stored, cloned or transferred.
The genes are checked using sequencing techniques and those with errors are rejected.

Question 14

What are the advantages of the gene machine?

Answer 14

Any sequence of nucleotides can be produced, in a short time and with great accuracy.
The artificial genes are also free of introns, and other non-coding DNA, so can be transcribed and translated by prokaryotic cells.

Question 15

How are the fragment genes cloned?

Answer 15

The fragments are cloned so there is sufficient quantity for medical or commerical use, by:
In vivo - by transferring the fragments to a host cell using a vector.
In vitro - using the polymerase chain reaction.

Question 16

What is the importance of sticky ends?

Answer 16

If the recognition sites are cut staggered, it leaves the ends of the DNA with a single strand, a few nucleotide bases long. These are complementary to the nucleotides on the other side.
If the same restriction endonuclease is used to cut DNA, then all the ends produced will have complementary ends, and the end can be joined to the end of another fragment.

Question 17

What is DNA ligase?

Answer 17

Once the complementary bases of two sticky ends have paired up, DNA ligase is used to bind the phosphate-sugar framework of the two sections of DNA, and so unite them as one.

Question 18

What is the preparation of the DNA fragment for insertion?

Answer 18

Additional lengths of DNA are added.
If we want the DNA fragment to transcribe mRNA and make a protein, it is essential to attach it to the necessary promoter region to start the process.
Similarly, a terminator region needs adding to the other end of the DNA fragment to stop transcription.

Question 19

What is the insertion of the DNA fragment into a vector?

Answer 19

The fragment of DNA, with promoter and terminator regions added, needs joining to the vector, which transports DNA to the host cell.
Most commonly used is a plasmid, circular lengths of DNA, in bacteria.
Plasmids usually contain genes for antibiotic resistance, and restriction endonucleases are used on this gene to break the plasmid loop.

Question 20

What do restriction endonucleases do for the insertion of DNA?

Answer 20

The restriction endonuclease used is the same one used to cut the DNA fragment.
This ensures that the sticky ends of the plasmid are complementary to the DNA fragment.
The DNA fragment can become incorporated into the plasmids, and joined permanently by DNA ligase.

Question 21

What is transformation?

Answer 21

The incorporated DNA in plasmids needs reintroducing to bacterial cells - transformation.
Plasmids and bacterial cells are mixed together in a medium containing calcium ions. Calcium, with temperature changes, make the bacterial membrane permeable, and allow plasmids to pass through the cell surface membrane into the cytoplasm.

Question 22

Why do not all the bacterial cells possess the DNA fragments with the desired gene?

Answer 22

Only a few bacterial cells take up the plasmid when the two are mixed.
Some plasmids will have closed up again without incorporating the DNA fragment.
Sometimes the DNA fragment ends join together to form its own plasmid.

Question 23

How are bacterial cells with the plasmid identified?

Answer 23

It uses the idea of bacteria’s evolved mechanisms of resisting the effects of antibiotics, by producing an enzyme that breaks down the antibiotic before it can destroy the bacterium.
The genes to produce these enzymes are found in plasmids.

Question 24

What are antibiotic resistant genes?

Answer 24

Some plasmids carry genes resistant to multiple antibiotics.
E.g. R-plasmid resists antibiotics ampicillin and tetracylcine.

Question 25

How are bacteria with the plasmids identified using ampicillin?

Answer 25

The gene for antibiotic resistance to ampicillin is unaffected by the introduction of the new gene.
All the bacterial cells are grown on a medium that contains ampicillin.
Bacterial cells that have taken up the plasmids will have the gene for ampicillin resistance.
They are able to break down ampicillin and so survive.

Question 26

What is the problem with antibiotic resistance as an identifier?

Answer 26

Some cells will have taken up the plasmids and then closed without incorporating the new gene, so will also have survived.
So the cells without the new gene need identifying and eliminating, using marker genes.

Question 27

What are marker genes?

Answer 27

Using marker genes to identify bacterial cells all involve using a second, separate gene on the plasmid, easily identifiable because:
It may be resistant to an antibiotic.
It may make fluorescent protein that is easily seen.
It may produce an enzyme whose action can be identified.

Question 28

What is replica plating?

Answer 28

Used to identify cells with plasmids with the new gene.
It uses the other antibiotic resistance gene in the plasmid, that was cut to incorporate the required gene.
The gene has been cut, so no longer produces the enzyme to break down the antibiotic - tetracycline.
These bacteria can then be identified by growing them on a culture with tetracycline.

Question 29

What is the limitation of using tetracycline?

Answer 29

The treatment with tetracycline will destroy the cells that contain the required gene.
However, by using replica plating, it is possible to identify the living colonies of bacteria with the required gene.

Question 30

What are fluorescent markers?

Answer 30

The transfer of a gene from a jellyfish into the plasmid, that produces a green fluorescent protein (GFP).
The gene to be cloned is transplanted into the centre of a GFP gene.
Any bacterial cell with the plasmid with the gene to be cloned will not produce GFP.

Question 31

What is the advantage of fluorescent markers?

Answer 31

The bacterial cells with the desired gene fluoresce, but do not die, so there is no need for replica plating.
Results can be obtained just by viewing the cells under a microscope, and retaining those that do not fluoresce, and is so more rapid.

Question 32

What are enzyme markers?

Answer 32

The gene that produces lactase can be a marker.
Lactase will turn a colourless substrate blue.
The required gene is transplanted into the gene of lactase.
If a plasmid with the required gene is present in the bacterial cell, the colonies will not produce lactase, so cannot change the substrate’s colour.
The cells that turn it blue can be discounted.

Question 33

What is the polymerase chain reaction?

Answer 33

PCR is used to copy fragments of DNA.
The process is automated, so is rapid and efficient.
The process requires the DNA fragment, DNA polymerase, primers, nucleotides, and a thermocycler.
The three stages are separation, addition, and synthesis.

Question 34

What is DNA polymerase?

Answer 34

An enzyme capable of joining together thousands of nucleotides in a few minutes.
Taq polymerase, is obtained from bacteria in hot springs and is therefore tolerant to heat (thermostable) and does not denature during the high temperatures used in PCR.

Question 35

What are primers?

Answer 35

Short sequences of nucleotides that have a set of bases complementary to those at one end of each of the two DNA fragments.

Question 36

What is a thermocycler?

Answer 36

A computer-controlled machine that varies temperatures precisely over a period of time.

Question 37

What is the separation of the DNA strand in PCR?

Answer 37

The DNA fragments, primers and DNA polymerase are placed in a vessel in a thermocycler.
The temperature is increased to 95°C, causing the two strands of the DNA to separate, due to the breaking of hydrogen bonds.

Question 38

What is addition of the primers in PCR?

Answer 38

The mixture is cooled to 55°C, causing the primers to join (anneal) to their complementary bases at the end of the DNA fragment.
The primers provide the starting sequences for DNA polymerase to begin DNA copying because DNA polymerase can only attach nucleotides to the end of an existing chain.
Primers also prevent the separated strands from rejoining.

Question 39

What is synthesis of DNA in PCR?

Answer 39

The temperature is increased to 72°C.
This is the optimum temperature for the DNA polymerase to add complementary nucleotides along each of the separated DNA strands.
It begins at the primer on both strands and adds the nucleotides in sequence until it reaches the end of the chain.

Question 40

What is the repetition of PCR?

Answer 40

Two copies of DNA are made from the original fragment.
The process is then repeated, resulting in 4 strands.
The cycle takes 2 minutes.
Over a million copies can be made in only 25 cycles, and 100 billion in a few hours.

Question 41

What is the application of PCR?

Answer 41

The tiniest sample of DNA from a single hair or speck of blood can be multiplied to allow forensic examination and accurate cross-matching.

Question 42

What are the advantages of in vitro gene cloning - time?

Answer 42

It is extremely rapid, which is particularly valuable when only a small amount of DNA is available.
This can be quickly increased by PCR, so there is no loss of time before forensic analysis and matching can take place.
However, PCR can also massively increase any other contaminating DNA found at a crime scene.

Question 43

What is the advantage of in vitro cloning - life?

Answer 43

It does not require living cells - only a base sequence of DNA that needs amplification.
No complex culturing techniques, requiring time and effort, are needed.

Question 44

What are the advantages of in vivo cloning - vectors?

Answer 44

It is particularly useful to add a gene into another organism.
It involves vectors, so once the gene is in the plasmid, the plasmid can add the gene to another organism, using gene therapy.

Question 45

What are the advantages of in vivo cloning - risk?

Answer 45

It involves little risk of contamination, because a gene that has been cut using the same restriction endonuclease is used, can match the sticky ends of the plasmid.
Contaminant DNA can not be taken up by the plasmid.

Question 46

What are the advantage of in vivo cloning - accuracy?

Answer 46

It is very accurate, with the copied DNA having very few errors.
At one time, about 20% of the DNA cloned by in vitro was copied inaccurately, but modern techniques have improved this.
However, any errors or contaminants will be copied.

Question 47

What are the advantages of in vivo cloning - specificity and yield?

Answer 47

It cuts out specific genes, so is very precise, as the culturing of transformed bacteria produces many copies of a specific gene, and not DNA as a whole.
It produces transformed bacteria that can be used to produce large quantities of gene products, used for commerical or medical use.