Chapter 21 - Recombinant Genes Flashcards
Steps of making recombinant DNA
1) isolation - isolating the fragments of DNA to be combined with another piece of DNA
in vivo cloning:
2) insertion
3) transformation
4) identification
5) growth/ cloning
What are the three methods to creates fragments of DNA (isolation step)
1) Using the enzyme Reverse transcriptase
2) Using the enzyme Restriction endonuclease
3) gene machine
Process of isolation using the enzyme reverse transcriptase
1) This enzyme makes DNA copies from mRNA (naturally occurs in viruses)
2) A cell that naturally processes the protein of interest is selected
3) These cells should have large amounts of mRNA for the protein
4) The reverse transcriptase enzyme joins the DNA nucleotides with complementary bases to the mRNA sequence
5) Single stranded DNA (cDNA) is made
6) to make this DNA fragment double stranded the enzyme DNA polymerase is used
Advantages of using reverse transcriptase for isolation
- the cDNA is intron free as it is based on the mRNA template
- mRNA present in cell is from actively transcribed genes, so lots of the mRNA of interest available to make cDNA
Process of restriction endonucleases for isolation
1) Restriction restriction endonuclease are enzymes that cut up DNA (occurs naturally in bacteria as a defence mechanism)
2) the enzyme has an active site complementary in shape with the DNA base sequences, known as recognition sequences
3) therefore the enzyme cuts the DNA at a specific location
4) some enzymes cut at the same location in the double strand and create a blunt end
5) other enzymes cut to create a staggered end and exposed DNA bases
6) the exposed staggered ends are palindromic (is the same if read backwards) and referred to as sticky ends as they have the ability to join to DNA with complementary base pairs
Process of gene machine for isolation
1) scientist first examine the protein of interests to identify the amino acid sequence and from that work out the mRNA and DNA sequence
2) The DNA sequence is entered into the computer which checks for bio saftey and bio security that the DNA being created is safe and ethical to produce
3) the computer can create short fragments of DNA (oligonucleotides)
4) the oligonucleotides can then be joined to create the DNA for the entire gene
5) PCR can be used to amplify the quantity and to make the double strand
advantage of gene machine
- the process is quick, accurate (100%)
- makes intron free DNA so can be transcribed in prokaryotic cells
- can design exact DNA fragment you want with sticky ends and preferential codons
disadvantages of reverse transcriptase
- more steps so more time consuming more technically difficult
disadvantages of restriction endonuclease
- still contains introns
disadvantage of gene machine
- need to know the sequence of amino acids or bases
advantages of restriction endonuclease
- sticky ends on DNA make it easier to insert to make recombinant DNA
Describe and explain what modifications need to be added to a gene before it can undergo insertion are (to ensure transcription can occur)
- promotor region - this is a sequence of DNA added to the start of the DNA fragment which acts as the binding site for RNA polymerase to enable transcription to occur
- terminator region - this is added to the end of the gene to cause RNA polymerase to detach and stop transcription, so only one gene at a time is copied into mRNA
what is a vector and example
something to carry the isolated DNA fragment into the host cell
e.g plasmid
Explain the process of insertion (inserting DNA into a vector)
1) The plasmid is cut open using the same restriction endonuclease
2) This creates the same sticky ends
3) Therefore the DA fragment, sticky ends are complementary to the sticky ends on the plasmid
4) The DNA fragment and cut plasmid are combined using the enzyme ligase
5) This occurs by ligase catalysing the condensation reaction to form phosphodiester bonds between nucleotides
Describe the process of transformation
1) the host cells are mixed with calcium ions and heat shocked increasing the permeability of the membrane
2) this enables the vector (plasmid with recombination DNA) to enter the host cells cytoplasm where the gene will be expressed to create the protein required
why doesn’t all the host cells successfully take up the recombinant plasmid
1) the recombinant plasmid doesn’t get inside the cell
2) the plasmid re-joins before the DNA fragment entered
3) the DNA fragments stick to itself rather than inserting into the plasmid
what are marker genes
genes on the plasmid used to identify which bacteria successfully took up the recombinant plasmid
state the different types of marker genes
1) antibiotic resistant genes
2) genes coding fo fluorescent proteins e.g green fluorescent protein (GFP)
3) genes coding for enzyme e.g enzyme lactase
Describe the antibiotic resistance method for identification
1) Genes for tetracycline and ampicillin antibiotic resistance are inserted into a bacterial plasmid
2) insert the isolated DNA fragment that we want to clone in the middle of the resistant tetracycline gene
3) this gene will no longer be able to translate a protein that will be resistant to tetracycline
4) then the bacteria is grown on agar
5) use a sterile velvet block to place over the Petri dish and transfer the bacterial colonies to a plate with ampicillin antibiotic in the agar
7) any colonies that can grow tells us it must have the plasmid inside of it as it has the gene making it resistant to ampicillin
8) use a sterile red velvet back to transfer these bacterial colonies that had grown to a plate with tetracycline antibiotic
9) colonies that grew on both Petri dishes is the original plasmid that does not contain gene of interest as it contains both ampicillin and tetracycline resistant genes
10) colonies that dont grow on tetracycline are the colonies that contain the recombinant plasmid
describe the process using GFP for identification
1) GFP is isolated and inserted into the bacterial plasmid
2) The DNA fragment that has been isolated is inserted into the middle of the GFP gene, this disrupts it and prevents GFP production
3) the bacteria is then grown on agar and exposed to UV light
4) The ones that contain the GFP gene fluoresce in UV light
5) the ones that dont glow contain the recombinant plasmid
what does the enzyme lactase do
turns substances colourless to blue
describe the process of the enzyme lactase for identification
1) the gene for the enzyme lactase is inserted into the plasmid
2) the DNA fragment is inserted in the middle of the lactase gene this disrupts it and prevents lactase production
3) the bacteria is then grown on an agar plate with a colourless substance
4) the colonies which cannot turn the colourless substance blue contain the recombinant plasmid
describe how the recombinant plasmid grows
1) a fermenter is used to grow multiple copies of the host cells which have been identified as containing recombinant plasmid
2) this large cloned population can then produce the protein coded by the inserted DNA fragment
Process of PCR
1) denaturing - the temperature is increased to 95 degrees to break the hydrogen bonds and split the DNA into single strands
2) annealing - the temperature is then decreased to 55 degrees so that primers can attach to the complementary bases at the end of the DNA
3) synthesis - the temperature is increased to the optimum temperature for taq DNA polymerase, 72 degrees, so that the taq DNA polymerase then attaches to complementary free nucleotides and add the nucleotides along the DNA strands until it reaches the end of the sequence
4) you will finish with two copies of the DNA fragment
