Section 8 - 21 Recombinant DNA technology Flashcards
What does recombinant DNA technology allow?
Genes to be manipulated, altered and transferred from organism to organism.
What is recombinant DNA?
Two different organisms that have been combined as a result of isolation, cloning and transferring.
State the stages involved in the transfer and cloning of DNA
- Isolation
- Insertion
- Transformation
- Identification
- Growth/clowing
What are three ways to perform the isolation stage of DNA technology?
- Conversion of mRNA to cDNA using reverse transcriptase
- restriction endonucleases to cut fragments containing the desired gene of DNA
- creating a gene using gene machine based on a known protein structure
How can reverse transcriptase be used in the isolation stage of DNA technology?
- select cell that readily produces the desired protein.
- these have large quantities of relevant mRNA - can be easily extracted.
- RT then used to make DNA from RNA.
- Complementary DNA (cDNA) is produced as nucleotides are complementary to mRNA.
- The enzyme DNA polymerase is used to build up te complementary nucleotides on the cDNA template.
How can restriction endonucleases be used for the isolation stage of DNA technology?
Each restriction endonuclease recognises and cuts DNA at a specific sequence of bases. - this specific site is called the recognition sequence.
- sticky ends - preferred
- smooth ends
How can the gene machine be used for the isolation stage of DNA technology?
- Determine desired amino acid sequence. - mRNA codon looked up and complementary DNA triplets worked out.
- Feed desired nucleotide bases into the computer.
- Check sequence for biosafety + biosecurity.
- Computer designs series of small, overlapping single strands of nucleotides.
- automated process, each oligonucleotide is assembled.
- Gene doesn’t have introns
- Can be replicated using polymerase chain reaction.
- sticky ends mean gene can e inserted into bacterial plasmid (vector)
What are the advantages of using the gene machine?
- any sequence of nucleotides can be produced.
- Quick (little as 10 days)
- Great accuracy.
- Free of introns.
In what two ways can you clone genes?
- in vivo - transfer into host cell using a vector
- in vitro - using polymerase chain reaction
Define - recognition site
The sequence of DNA that is cut by restriction endonucleases.
What is the enzyme DNA ligase used for?
Once the complementary bases of two sticky ends have been paired up. DNA ligase used to bind the phosphate-sugar framework of the two sections of DNA and so unite them as one.
Why are sticky ends so important?
- provide the same restriction endonuclease is used
- we can combine the DNA of one organism with that of any other organism.
How is a DNA fragment prepared for insertion?
- RNA polymerase attaches to the DNA near a gene
- Promotor - the biding site for RNA polymerase.
- The nucleotide bases of the promotor attach both RNA polymerase and transcription factors.
- This begins the process of transcription.
- At the same time the terminator region releases RNA polymerase and ends transcription.
Why is plasmid the most common type of vector used?
- Circular length of DNA
- found in bacteria
- contain genes from antibiotic resistance
- the restriction endonuclease can be used at one of these genes to break the plasmid loop.
Why is the same restriction endonuclease used during isolation and insertion?
- ensuring the sticky ends of the opened-up plasmid are complementary to the sticky ends of the DNA fragment.
Explain the process of insertion
- break plasmid at antibiotic resistance site.
- Use same restriction endonucleases.
- When the fragment is mixed with opened up plasmid they become incorporated
- join made permanently using DNA ligase
- Now known as recombinant DNA
What does transformation involve?
- plasmid and bacterial cells being mixed together in a medium containing calcium ions.
- The calcium ions and change in temp make bacterial membrane permeable.
- Plasmids can pass through into cytoplasm.
Why don’t all bacterial cells possess the DNA fragment after insertion?
- fewer than 1% of bacterial cells take up plasmids when mixed.
- some plasmids will close without incorporating fragment
- DNA fragment may join together to form own plasmid