21.1 Producing DNA fragments Flashcards
What is recombinant DNA
Isolate genes, clone them and transfer them to microorganisms.
These microorganisms then grow to form a factory for the continuous production of a desired protein
If a person isn’t able to produce chemicals that are proteins, eg insulin this technology can be used.
What is the resulting organism of recombinant DNA called
A transgenic or genetically modified organism
How is it possible that the DNA of one organism is transferred to another and can function normally
Genetic code is the same in all organisms: It is universal and can be used by all living organisms.
So the DNA can be interpreted, and transcription/translation mechanisms are essentially the same in all organisms so proteins can be made too.
So as a result transferred DNA can be transcribed and translated to product the required protein
What are the stages of making a protein using DNA technology of gene transfer and cloning
1) Isolation of DNA fragments that have the gene for the required fragments
2) Insert DNA fragment into a vector
3) Transfer the DNA into suitable host cells
4) Use gene markers to identify the host cells that took up the gene
5) Growth/cloning of the population of host cells
What must happen to a gene before it is transplanted
It must be identified and isolated from the rest of the DNA
The required gene may consist of a sequence of a few hundred bases out of the millions in DNA.
So there are a few methods of DNA fragmentation
3 Methods of DNA fragmentation
Reverse transcriptase can convert mRNA to cDNA
Use restriction of endonucleases to cut fragments containing the desired gene from DNA
Create gene in a gene machine
How do you use reverse transcriptase to fragment genes
In a host cell they’re able to synthesise DNA from RNA
1) B cells from Islets of Langerhans in the pancreas produce insulin so contain lots of mRNA that codes for it
2) Remove mRNA and this acts as template strand where complimentary DNA can be made (cDNA) is formed. This is called complimentary DNA.
3) cDNA is isolated by hydrolysis of the mRNA with an enzyme
4) Then this single strand of cDNA is turned into double stranded DNA by DNA polymerase
5) Copy of human insulin gene is made
How do we use restriction endonucleases to fragment genes
(Recognition sequence)
- Bacteria are infected by pathogens eg viruses that insert their DNA into them to take over the cell
- Some bacteria protect themselves by producing enzymes that cut up viral DNA.
These enzymes are called restriction endonucleases:
How do restriction endonucleases cut DNA double strand
What is a palindrome
This cut can occur…
Between two opposite base pairs: This leaves straight edges called blunt ends
It can also occur in a staggered fashion, and leaves uneven cut so each strand of DNA has exposed base pairs.
If you read both sequences from left to right they are opposites of each other so are called palindromes
How does the gene machine work to manufacture genes
. Look at protein we want to produce, and we can decide the desired nucleotides we need to make it. Determine amino acid sequence of protein.
. From this the mRNA codons are looked up and complimentary DNA triplets are worked out
. Desired sequence of nucleotide bases for the gene is fed into the computer
. Sequence is checked for biosafety and biosecurity so it reaches international standards and ethical ones
. Computer makes oligonucleotides which are small overlapping single strands of nucleotides
. Oligonucleotides are assembled by adding one nucleotide at a time in the required sequence
. Then add oligonucleotides to join them into a gene which has no introns
. Replicate gene in polymerase chain reaction which also makes the other complementary strand of nucleotides to make double stranded gene
. Then use sticky ends to insert gene into bacterial plasmid which acts as vector for gene so it can be stored, cloned or transferred to organism in the future
Advantages of gene machine
. No introns made so prokaryotic cells can transcribe and translate it
. Any sequence of nucleotides can be produced in a very short time and great accuracy
