Recombinant DNA Technology Flashcards
Stages of Genetic Fingerprinting
- Extraction
- Digestion
- Separation
- Hybridisation
- Development
Extraction during genetic fingerprinting
DNA is extracted by phenol and chloroform, separating it from the rest of the cell.
The quantity of DNA is increased by the polymerase chain reaction
Digestion during genetic fingerprinting
DNA is cut into fragments by restriction endonucleases.
Restriction endonucleases are chosen because they cut close to, but not within, the target DNA
Separation during genetic fingerprinting
Fragments of DNA are separated according to size by gel electrophoresis
The gel is immersed in alkali to separate the double strands into single strands
Hybridisation during genetic fingerprinting
DNA probes (radioactive/fluorescent) bind with VNTR
The probes have complementary base sequences to the VNTRs
The process uses different probes to bind with different DNA sequences
Development of genetic fingerprinting
An x-ray film is out over the nylon membrane
The film is exposed to the radiation from the radioactive probes, or from fluorescent probes by which light is visually identified
These point correspond to the position of the DNA fragments as separated during electrophoresis, so a series of bars is revealed.
Methods of producing DNA fragments
- Using Reverse Transcriptase
- Using Restriction Endonucleases
- Using the Gene Machine
Using Reverse Transcriptase to produce DNA fragments
- Cell that readily produces lots of a protein is selected due to a high amount of mRNA
- mRNA acts as a template which reverse transcriptase acts upon, forming cDNA
- cDNA is isolated by hydrolysis
- Double stranded DNA is formed on the cDNA template by DNA polymerase. The double stranded DNA is the gene
Using Restriction Endonucleases to produce DNA fragments
- Restriction Endonucleases cut the DNA strand at a specific sequence known as a recognition site.
- Can cut between two opposite base pairs producing blunt ends, or in the middle of the site producing sticky ends
Using the ‘Gene Machine’ to produce DNA fragments
- Desired amino acid sequence of the protein is determined, the mRNA codons are looked up and the complementary DNA triplets are worked out.
- The desired nucleotide sequence is fed into a computer where it is checked for biosafety concerns.
- The computer designs small, overlapping single strands of nucleotides, called oligonucleotides that can be assembled into the desired gene
- In an automated process, each of the oligonucleotides are assembled one at a time in the required sequence.
- The oligonucleotides are then joined together to make a gene. The gene has no introns and is replicated by the polymerase chain reaction.
- The polymerase chain reaction constructs the complementary strand of nucleotides to make the required double stranded gene. Using sticky ends, the gene is inserted into a bacterial plasmid allowing it to be cloned and errors are rejected.
Advantages: Quick, accurate and no introns.
Three Stages of the Polymerase Chain Reaction
- Separation (95 Degrees)
- Addition of primers (55 Degrees)
- Synthesis of dna (72 Degrees)
Components needed for the polymerase chain reaction
- DNA Nucleotides - to be added to form the cloned fragment
- DNA fragment(s) to be cloned
- Primers - to begin the addition of nucleotides, have base sequences complementary to the start and end of the fragment
- DNA polymerase - to add nucleotides
- Thermocycler - hold the mixture and change the heat accordingly
First stage of the Polymerase chain reaction (95)
The thermocycler is heated to 95 degrees to break the hydrogen bonds between the DNA fragment, exposing the nucleotides
Second stage of the Polymerase chain reaction (55)
The thermocycler is heated to 55 degrees because it is the optimum temperature for primers to be added to either end of the DNA fragment. The primers allow the addition of nucleotides to begin
Third stage of the Polymerase chain reaction (72)
The thermocycler is heated to 72 degrees because it is the optimum temperature for DNA polymerase to act on the separated strand, joining nucleotides together and forming two new DNA fragments