amplifying DNA fragments

Question 1

Fragments of DNA can be amplified by

Answer 1

in vitro and in vivo techniques

Question 2

what is the main in vitro method?

Answer 2

PCR (polymerase chain reaction)

Question 3

what is the main in vivo method?

Answer 3

transferring the fragments to a host cell using a vector

Question 4

why are the ‘sticky ends’ produced from restriction endonucleases important for in vivo amplification?

Answer 4

considerable importance because provided the same restriction enzyme is used (so same recognition sites are cut) we can combine the DNA of one organism with that of any other organism- once the complementary bases of the two ‘sticky ends’ have paired up an enzyme called DNA ligase is used to bind the phosphate-sugar framework of the two DNA sections to combine them as one

Question 5

what are the main steps of in vivo amplification?

Answer 5

1- preparing DNA fragments for insertion
2- insertion of DNA fragment into a vector
3- introduction of DNA into host cells
4- marker genes

Question 6

explain step 1 of in vivo amplification: preparding DNA fragment for insertion

Answer 6

• for transcription of any gene to take place RNA polymerase must attach to the DNA near a gene= promoter
• the nucleotide bases of the promoter attach both RNA polymerase and transcription factors= translation can begin
• so if we want our DNA fragment to transcribe mRNA in order to make a protein it is VITAL that it is attached to the necessary promoter region in order to start the process
• terminator region also needs to be added to other end of DNA fragment to stop transcription at the appropriate point

Question 7

explain step 2 of in vivo amplification: insertion of DNA fragment into a vector

Answer 7

1- once an appropriate fragment of DNA has been cut from the rest of the DNA and the promoter and terminator regions added the next task is to add it to a carrying unit- a vecto, used to transport DNA into a host cell
2- most common type of vector used is the plasmid (circular lenghts of DNA found in bacteria that are separate from the main bacterial DNA). Plasmids most always contain genes for antibiotic resistance and restriction enzymes are used at one of these antibiotic resistance genes to break the plasmid loop
3- the restriction enzyme used is the same on that cut out the DNA fragment= ensures that sticky ends of the opened up plasmid are complementary to the sticky ends of the DNA fragment
4- when the DNA fragments are mixed with the opened-up plasmids, they may become incorporated into them- when they are incorporated the join is made permanent by using DNA ligase
5- these plasmids now have recombined DNA

Question 8

regarding introduction of DNA into host cells, once the DNA has been incorporated into at least a few plasmids, they must then be reintroduced into bacteria cells- a process called

Answer 8

transformation

Question 9

what does transformation involve?

Answer 9

• plasmids and bacteria cells being mixed together in a medium containing calcium ions- calcium ions and change in temperature make the bacterial membrane permeable allowing the plasmids to go through the cell-surface membrane into the cytoplasm

Question 10

after transformation, why may not all the bacterial cells possess the DNA fragments with the desired gene for desired protein; some reasons ma be:

Answer 10

1- only a few bacterial cells, as few as 1% take up the plasmids when the two are mixed together
2- some plasmids will have closed up again without incorporating the DNA fragment

Question 11

the task of finding out which bacterial cells have taken up the plasmids entails using the gene for antibiotic resistance which will be unaffected by the introduction of the new gene- process works as follows:

Answer 11

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

Question 12

after discovering which bacterial cells have taken up the plasmids, some cells will have taken up the plasmid and then closed up without incorporating the new gene which will also have survived, so the next task is to

Answer 12

identify the new cells without the new gene and eliminate them achieved using marker genes

Question 13

there are a number of different ways of using marker genes to identify whether a gene has been taken up by the bacterial cells; they all involve the use of a second, seperate gene on the plasmid. This second gene is easily identifiable for one reason or another:

Answer 13

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

Question 14

explain use of antibiotic-resistance marker genes

Answer 14

• rather old method and been suspended by other methods
• to identify those cells with plasmids that have taken up the gene of interest a technique called replica plating is used
• the process uses the other antibiotic-resistance gene: the gene that was cut in order to incorporate the required gene e.g. resistance to tetracycline
• as this gene has been cut, it will no longer produce enzyme that breaks down tetracycline
• we can therefore identify these bacteria by growing them on a culture that contains tetracycline

Question 15

explain how replica plating works

Answer 15

• take an absorbent cloth such as sterile velvet marked N,E,S,W and place onto the petri dish containing the bacteria (only some of the bacteria have taken up the gene of interest however) in the same orientation
• this will cause some of the bacteria to be transferred to the cloth which can then be placed onto tetracycline medium
• the ones that die therefore must have taken the gene of interest and so you can take those cultures from the original plate and use them to extract the protein produced from the gene of interest

Question 16

explain use of fluorescent markers

Answer 16

• more recent and more rapid method is 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 cente of the GFP gene
• any bacterial cell that has taken up the plasmid with the gene of interest will not be able to produce GFP so bacterial cells that have NOT taken up the gene of interest will continue to produce GFP and fluoresce

Question 17

what makes process of using fluorescent markers more rapid than use of antibiotic-resistance marker gene

Answer 17

• bacterial cells with desired gene not killed so no need for replica plating
• results can simply be obtained by simply viewing the cells under a microscope and retaining those that do not fluoresce = rapid process

Question 18

explain use of enzyme markers

Answer 18

• another gene marker is the gene that produces the enzyme lactase
• lactase will turn a particular colourless substrate blue
• gene of interest is transplanted into the gene that makes lactase
• if a plasmid with the gene of interest is present in the 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

Question 19

define vector

Answer 19

simply a carrier- the term may refer to something like a plasmid which carries DNA into a cell

Question 20

what does PCR do?

Answer 20

method of copying fragments of DNA (process=automated so is rapid and efficient)

Question 21

what does PCR require?

Answer 21

• DNA fragment to be copied
• taq polymerase (obtained from bacteria in hot springs so is thermostable and does not denature at high temps)
• primers
• nucleotides
• thermocycler (a computer-controlled machine that varies temps precisely over a period of time)

Question 22

what are primers?

Answer 22

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

Question 23

what are the 3 main stages of PCR?

Answer 23

1- separation of DNA strand
2- addition (annealing of the primers)
3- synthesis of DNA

Question 24

explain what happens in the first stage of PCR (separation of DNA strand)

Answer 24

• DNA fragments, primers and DNA polymerase are placed into a vessel in thermocycler
• temp is increased to 95 degrees celcius
• causing the two strands of DNA fragment to separate due to breaking of hydrogen bonds between 2 DNA strands

Question 25

explain what happens in second stage of PCR (annealing of primers)

Answer 25

• mixture is cooled to 55 degrees celcius causing the primers to join (anneal) to their complementary bases at the end of the DNA fragment
• primers provide the starting sequence for DNA polymerase to begin DNA copying because DNA polymerase can only attach nucleotides to the end of an existing chain
• primers also prevent 2 strands rejoining

Question 26

explain what happens in third stage of PCR (synthesis of DNA)

Answer 26

• temp increased to 72 degrees celcius
• this is the optimum temperature for the DNA polymerase to add complementary nucleotides along each of the separate DNA strands
• it begins at primer on both strands and adds the nucleotides in sequence until it reaches the end of the chain

Question 27

in PCR, because both strands are copied simultaneously

Answer 27

• there are now 2 copies of original fragment
• once the 2 DNA strands are completed, the process is repeated by subjecting to them to the temperature cycle again, resulting in 4 strands
• whole temp cycle takes around 2 mins
• over a million copies of the DNA can be made in 25 temp cycles

Question 28

what are the advantages to in vitro cloning?

Answer 28

• extremely rapid= - over a million copies of the DNA can be made in 25 temp cycles
• so even tiniest sample of DNA from a single hair or speck of blood can now be multiplied to allow forensic examination and cross-matching which would take weeks to produce same amount via in vivo
• does not require living cells = no complicated/time consuming culturing techniques

Question 29

what are the advantages of in vivo cloning?

Answer 29

• particularly useful where we wish to introduce a gene into another organism- used in gene therapy
• involves almost no risk of contamination as a gene cut by same restriction enzyme can match sticky ends on plasmid =contaminant DNA not taken up into plasmid
• very accurate as mutations are very rare
• produces transformed bacteria that can be used to produce large quantities of gene products for commercial or medical use e.g insulin

Question 30

what are some benefits of recombinant DNA technology?

Answer 30

• microorganisms can be modified to produce a range of substances for example antibiotics, hormones and enzymes used to treat diseases and disorders
• genetically modified crops can help prevent certain diseases- a type of rice called golden rice can have a gene for vitamin A production added. Can we justify not developing more vitamin A enriched crops hundreds of millions of children worldwide at at risk from vitamin A deficiency leading to cases of irreversible blindness each year
• replacing defective genes (gene therapy) might be used to cure certain genetic disorders such as cystic fibrosis
• genetic fingerprinting can be used in forensic science

Question 31

what are some risks of recombinant DNA technology?

Answer 31

• will knowledge of and ability to change, human genes lead to eugenics whereby selection of genes leads to a mans of selecting one race over the other
• what will the consequences of the ability to manipulate genes getting into wrong hands? Will groups or governments use this power to achieve political goals or gain ultimate power?
• what will be the long-term consequences of introducing new gene combinations? We cannot be sure of the future events on the future evolution of organisms. Will the artificial selection of ‘desired’ genes reduce the genetic diversity essential to evolution
• any manipulation of the DNA of a cell will have consequences for the metabolic pathways within that cell- we cannot be sure until after the event what unforseen by-products of the change may be produced. Could these lead to metabolic malfunctions, cause cancer or create a new form of disease?
• financial consequences of developing plants to grow in new regions?