Gene technologies

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

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

Why does recombinant DNA technology work?

Answer 4

Because the genetic code is universal, and therefore transcription and translation occur by the same mechanism and result in the same amino acid sequence across organisms

Question 5

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

Answer 5

• 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 6

What are the methods of producing DNA fragments?

Answer 6

• 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 7

What is the process of using reverse transcriptase to produce DNA fragments?

Answer 7

mRNA complementary to the target gene is used as a template. It is mixed with free nucleotides which match up to their base pairs, and reverse transcriptase which forms the sugar-phosphate backbone, to create cDNA

Question 8

What are retroviruses?

Answer 8

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

Question 9

How is reverse transcriptase used to isolate a gene?

Answer 9

• A cell that readily produces the protein is selected. (E.g. B cells pf 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 10

How do bacteria use restriction endonucleases?

Answer 10

• 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 11

What are restriction endonucleases?

Answer 11

• 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 12

What do other types of restriction endonucleases do?

Answer 12

• 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 13

What is the process of using enzymes to produce DNA fragments?

Answer 13

• Restriction endonucleases cut DNA at specific sequences
• Different REs cut at different points but one RE will always cut at the same sequence
• therefore using particular REs allows you to cut out a certain gene of interest

Question 14

How does the gene machine work?

Answer 14

• 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 15

How does the gene machine work - oligonucleotides?

Answer 15

• 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 multiple to give many copies.

Question 16

How does the gene machine work - insertion?

Answer 16

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 checking using sequencing techniques and those with errors are rejected.

Question 17

What are the advantages of the gene machine?

Answer 17

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 18

How can we amplify/clone DNA fragments?

Answer 18

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

Question 19

What is the importance of sticky ends?

Answer 19

• 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, the all the ends produced will have complementary ends, and the end can be joined to the end of another fragment.

Question 20

What is DNA ligase?

Answer 20

• 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.
  Sticky ends mean we can combine the DNA of one organism with that of another organism.

Question 21

What is the preparation of the DNA fragment for insertion?

Answer 21

• 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 22

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

Answer 22

• 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 23

What do restriction endonucleases do for the insertion of DNA?

Answer 23

• the same one is used as the one that cut out the DNA fragment
• this ensures that the sticky ends of the opened-up plasmid are complementary to the sticky ends of the DNA fragment
• When the DNA fragments are mixed with the opened-up plasmids, they may become incorporated into them
• where they are incorporated, the join is made permanent using the enzyme DNA ligase
• these plasmids now have recombinant DNA

Question 24

What is transformation?

Answer 24

• when the DNA has been incorporated into at least some of the plasmids and reintroduced into bacterial cells
• it involves the plasmids and bacterial cells being mixed together in a medium containing calcium ions
• These ions and changes in temp, make the bacterial membrane permeable, allowing plasmids to pass through the cell-surface membrane into the cytoplasm

Question 25

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

Answer 25

- only a few bacterial cells (as few as 1%) take up the plasmids when the two are mixed together - 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 26

How are bacterial cells with the plasmid identified?

Answer 26

- use the fact that bacteria have evolved mechanisms for resisting the effect of antibiotics, typically by producing an enzyme that breaks down the antibiotic before it can destroy the bacterium

Question 27

What are antibiotic resistant genes?

Answer 27

- some plasmids carry genes for resistance to more than one antibiotic - e.g. the R-plasmid carries genes for resistance to 2 antibiotics, ampicillin and tetracycline

Question 28

How are bacteria with the plasmids identified using ampicillin?

Answer 28

- all the bacterial cells are grown on a medium that contains the antibiotic ampicillin - bacterial cells that have taken up the plasmids will have acquired the gene for ampicillin resistance - these bacterial cells are able to break down the ampicillin and therefore survive - the bacterial cells that have not taken up the plasmids will not be resistant to ampicillin and therefore die

Question 29

What is the problem with antibiotic resistance as an identifier?

Answer 29

- some cells will have taken up the plasmids and then closed up without incorporating the new gene, and these will also have survived

Question 30

What are marker genes?

Answer 30

- they can be used to identify whether a gene has been taken up by bacterial cells - they involve using a second gene on the plasmid

Question 31

How can the second gene in marker genes be indentified?

Answer 31

- it may be resistant to an antibiotic - it may make a fluorescent protein that is easily seen - it may produce an enzyme whose action can be identified

Question 32

What are antibiotic-resistance maker genes?

Answer 32

- the use of these as markers is an old technology and has been superseded by other methods - its an example of how scientists use knowledge and understanding to solve new problems, use appropriate methodology and carry out relevant experiments

Question 33

What is replica plating?

Answer 33

- uses other antibiotic-resistance gene in the plasmid: the gene that was cut in order to incorporate the required gene - as the gene has been cut it will no longer produce the enzyme that breaks down tetracycline - the bacteria that have taken up the required gene will no longer be resistant to tetracycline - therefore we identify these bacteria by growing them on a culture that contains tetracycline

Question 34

What is the limitation of using tetracycline?

Answer 34

- it will destroy the cells that contain the required gene - However, by using replica plating it is possible to identify living colonies of bacteria containing the required gene

Question 35

What are fluorescent markers?

Answer 35

- the transfer of a gene from a jellyfish into the plasmid - the gene in question produces a green fluorescent protein - the gene to be cloned is transplanted into the centre of the GFP gene - any bacterial cell that has taken up the plasmid with the gene that is to be cloned will not be able to produce GFP - those that have not will continue to produce GFP and to fluoresce - as the bacterial cells with the desired genes are not killed there's no need for replica plating

Question 36

What is the advantage of fluorescent markers?

Answer 36

- results can be obtained by simply viewing the cells under a microscope and retaining those that do not fluoresce - make the process more rapid

Question 37

What are enzyme markers?

Answer 37

- genes that produces the enzyme lactase - lactase will turn a particular colourless substrate blue - the required gene is transplanted into the gene that makes lactase - if a plasmid with the required gene is present in a bacterial cell, the colonies grown from it will not produce lactase - therefore when these bacterial cells are grown on the colourless substrate they will be unable to change its colour - where the gene has not been taken up by the bacteria, they will not turn the substrate blue - these bacteria can be discounted

Question 38

What is the polymerase chain reaction?

Answer 38

- a method of copying fragments of DNA - the process is automated, making it both rapid and efficient

Question 39

What is DNA polymerase?

Answer 39

- an enzyme capable of joining together tens of thousands of nucleotides in a matter of minutes - one such enzyme, taq polymerase, is obtained from bacteria in hot springs and is therefore tolerant to heat and does not denature during the high temps used as part of the process

Question 40

What are primers?

Answer 40

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

Question 41

What is a thermocyclers?

Answer 41

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

Question 42

What are the 3 stages of the PCR chain reaction?

Answer 42

1. separation of the DNA strand 2. addition of the primers 3. synthesis of DNA

Question 43

What is the separation of the DNA strand in PCR?

Answer 43

- the DNA fragments, primers and DNA polymerase are placed in a vessel in the thermocycler - the temperature is increased to 95°C, causing 2 strands of the DNA fragments to separate due to the breaking of the hydrogen bonds between the 2 DNA strands

Question 44

What is addition/annealing of the primers in PCR?

Answer 44

- the mixture is cooled to 55°C, causing the primers to 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 2 separate strands from simply rejoining

Question 45

What is synthesis of DNA in PCR?

Answer 45

- the temperature is increased to 72°C - this is the optimum temp 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 46

What is the repetition of PCR?

Answer 46

- because both separated strands are copied simultaneously there are now 2 copies of the original fragment - once the 2 DNA strands are completed, the process is repeated by subjecting them to the temperature cycle again, resulting in 4 strands

Question 47

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

Answer 47

- it is extremely rapid - within a matter of hours 100 billion copies of a gene can be made - this is particularly valuable where only a minute amount of DNA is available - this can quickly be increased using the polymerase chain reaction and so there is no loss if valuable time before forensic analysis and matching can take place - a complicating factor is that PCR will also increase massively any other contaminating DNA found at the scene - In vivo cloning would take many weeks to produce the same quantity of DNA

Question 48

What is the advantage of in vitro cloning - life?

Answer 48

- it doesn't require living cells - all that is required is a base sequence of DNA that needs amplification - no complex culturing techniques, requiring time and effort are needed

Question 49

What are the advantages of in vivo cloning - vectors?

Answer 49

- it is particularly useful where we wish to introduce a gene into another organism - it involves vectors, once the gene is introduced into a plasmid, the plasmid can be used to deliver the gene into another organism - gene therapy

Question 50

What are the advantages of in vivo cloning - risk?

Answer 50

- it involves almost no risk of contamination - because a gene that has been cut by the same restriction endonuclease can match the sticky ends of the opened-up plasmid - contaminant DNA will therefore not be taken up by the plasmid - In Vitro cloning requires a very pure sample because any contaminant DNA will also be multiplied and could lead to false result

Question 51

What are the advantage of in vivo cloning - accuracy?

Answer 51

- very accurate - The DNA copied has very few errors - modern techniques have improved this - However, any errors in copying DNA or any contaminants in the sample will also be copied in subsequent cycles - very rare that mutations will happen

Question 52

What are the advantages of in vivo cloning - specificity?

Answer 52

- it cuts out specific genes - therefore very precise procedure as the culturing of transformed bacteria produces many copies of a specific gene and not just copies of the whole DNA sample

Question 53

What are the advantages of in vivo cloning - yield?

Answer 53

- it produces transformed bacteria that can be used to produce large quantities of gene products - the transformed bacteria can produce proteins for commercial or medical use e.g. hormones such as insulin