Recombinant DNA technology Flashcards
What is recombinant DNA technology?
Involves the transfer of fragments of DNA from one organism or species to another. It can enable scientists to manipulate + alter genes to improve industrial processes + medical treatment
First step is to produce or isolate the fragments of DNA to be recombined with another piece of DNA
3 methods:
- using reverse transcriptase
- using restriction enzymes
- using gene machine
Reverse transcription
- reverse transcriptase makes DNA copies from mRNA
- naturally occurs in viruses such as HIV
- a cell that naturally produces the protein of interest is selected
- these cells should have large amounts of mRNA for the protein
- the reverse transcriptase enzyme joins the DNA nucleotides with complementary bases to the mRNA sequence
- single stranded DNA is made (cDNA)
- to make DNA fragment double stranded, DNA polymerase is used
Advantage: cDNA is intron free because it’s based on mRNA template
Restriction endonucleases
- They are enzymes that cut up DNA + they naturally occur in bacteria as a defence mechanism
- There are many restriction enzymes that have an active site complementary in shape to a range of different DNA base sequences (recognition sequences) + so each enzyme cuts DNA at a specific location
- Some enzymes cut at the same location in the double strand + create a blunt end, other enzymes cut to create staggered ends + exposed DNA bases
- The exposed staggered ends are palindromic (same forwards as it is backwards) and are called sticky ends as they have the ability to join to DNA with complementary base pairs
Gene machine
- DNA fragments can be created in a lab with a computerised machine
- Scientists first examine the protein of interest to identify the amino acid sequence + from that work out what the mRNA + DNA base sequence would be
- DNA sequence is entered into a computer which checks for biosafety+ biosecurity that the DNA created is safe + ethical to produce
- The computer can create small sections of overlapping single strands of nucleotides that make up the gene, called oligonucleotides
- Oligonucleotides can then be joined to create the DNA for the entire gene
- PCR can be used to amplify the quantity + make the double strand
- This process is very quick, accurate + makes intron free DNA so can be transcribed in prokaryotic cells
Advantages + disadvantages of reverse transcription
+ : using a cell which creates the protein naturally so lots of mRNA present in that cell to make lots of copies of cDNA
- : more steps so more time consuming + more difficult
Advantages + disadvantages of restriction endonucleases
+ : sticky ends on DNA fragment make it easier to insert to make recombinant DNA
- : still contains introns
Advantages + disadvantages of gene machine
+ : can design exact DNA fragment you want with sticky ends + labels
- : need to know the sequence of amino acids or bases
Stages of in vivo gene cloning
1) Desired gene is isolated using restriction endonuclease enzymes
2) Promoter regions + terminator regions are added to the gene to make sure the gene can be correctly transcribed once in the host
3) The gene is inserted into a vector (plasmid)
4) The recombinant plasmid is now transferred to host cells
5) The host cells are allowed to multiply + those that have successfully taken up the gene are identified using a marker gene
State the modifications of the DNA fragments
DNA fragments must be modified to ensure transcription of these genes can occur
Promotor region added - added at start of DNA fragment + is a sequence of DNA which is the binding site for RNA polymerase to enable transcription to occur
Terminator region added - added at end of gene + causes RNA polymerase to detach + stop transcription, so only one gene at a time is copied into mRNA
Process of inserting DNA into a vector
A vector is something to carry the isolated DNA fragment into the host cell (plasmids are most common vectors)
1) Plasmid is cut open using the same restriction endonuclease enzyme
2) This creates the same sticky ends
3) The DNA fragment sticky ends are complemetary to the sticky ends on the plasmid
4) The DNA fragment + cut plasmid are combined + enzyme ligase sticks them together. Ligase catalyses the condensation reaction to form phosphodiester bonds between nucleotides
How are vectors (plasmids) transferred into host cells?
- The vectors needs to be inserted into the host cell where the gene will be expressed to create the protein required
- The cell membrane of the host cell must be more permeable
- To increase permeability, host cells are mixed with ca2+ and heat shocked (sudden increase in temp)
- This enables the vector to enter the host cell’s cytoplasm
Identifying transformed cells
Scientists must identify which bacteria successfully took up a recombinant plasmid.
Not all host cells with successfully take up the recombinant plasmid
3 issues can occur:
- the recombinant plasmid doesn’t get inside the cell
- the plasmid rejoins before the DNA fragment entered
- the DNA fragment sticks to itself rather than inserting into the plasmid
3 different marker genes
used to identify which bacteria successfully took up the recombinant plasmid
- Antibiotic resistance genes
- Genes coding for fluorescent proteins
- Genes coding for enzymes
Antibiotic resistance marker genes
- make sure you insert a gene that makes the bacteria resistant to tetracycline + the gene to make it resistant to ampicillin
- then insert DNA fragment into plasmid
- resistance to tetracycline gene is disrupted by DNA fragment so gene will no longer create a functional protein
- Grow bacteria on agar
- Transfer bacterial colonies to a plate with ampicillin antibiotic in agar
- Transfer bacterial colonies to a plate with tetracycline antibiotic in agar
- So can identify the bacteria containing the required gene
Fluorescent markers
- Jellyfish contain a gene which codes to create a green fluorescent protein (GFP)
- This GPF gene can be inserted into the bacteria plasmid
- DNA fragment is inserted into middle of GFP gene. This disrupts it + prevents GFP production
- Grow bacteria on agar + if expose colonies to UV light, the non-glowing colonies contain the recombinant plasmid
