Recombinant DNA technology Flashcards
What is recombinant DNA
The DNA of two different organisms that has been combined
What is a transgenic or genetically modified organism
One which contains recombinant DNA.
What is the key reason that genetic recombinant technology works
The DNA code is universal- it is the same in all organisms
What are the five stages in making a protein using the DNA technology of gene transfer and cloning
1) ISOLATION of the DNA fragments that have the gene for the desired protein.
2) INSERTION of the DNA fragment into a vector.
3) TRANSFORMATION: the transfer of DNA into suitable host cells.
4) IDENTIFICATION of the host cells that have successfully taken up the gene by use of gene markers
5) GROWTH/CLONING of the population of host cells.
What are the three methods for producing DNA fragments that you need to know
1) Conversion of mRNA to cDNA using reverse transcriptase
2) Using restriction endonucleases to cut fragments containing the desired gene from DNA
3) Creating the gene in a gene machine, usually based on a known protein structure.
What does the enzyme reverse transcriptase do
Catalyse the production of DNA from RNA.
Describe generally how DNA fragments are made using reverse transcriptase
- A cell that readily produces the desired protein is selected.
- These cells have large quantities of the relevant mRNA, which is therefore more easily extracted
- Reverse transcriptase is then used to make DNA from RNA.
- This DNA is known as complementary DNA (cDNA) because it is made up of the nucleotides that are complementary to the mRNA.
- To make the other strand of DNA, the enzyme DNA polymerase is used to build up to complementary nucleotides on the cDNA template.
- This double strand of DNA is the required gene.
Describe how reverses transcriptase is used to isolate the gene that codes for insulin
- B cells from the islets of Langerhans in the human pancreas are selected as they are specialised to produce insulin so make a lot of mRNA that codes for insulin.
- The mRNA that codes for insulin is extracted
- The mRNA acts as a template on which a single-stranded complementary copy of DNA (cDNA) is formed using reverse transcriptase .
- Single-stranded cDNA is isolated by hydrolysis of the mRNA with an enzyme.
- Double stranded DNA is formed on the template of the cDNA using DNA polymerase.
- A copy of the human insulin gene has now been produced.
What are restriction endonucleases
Enzymes produced by bacteria to defend against viral infections- they do this by cutting up the viral DNA.
What are the two ways that restriction endonucleases cut a strand of DNA
1) Leaving blunt ends by cutting in a straight line between two opposite base pairs.
2) Leaving sticky ends by cutting the DNA in a staggered fashion.
What is a recognition sequence
The specific sequence of DNA bases where a type of endonuclease cuts the DNA .
What is the common type of recognition sequence for restriction endonucleases that cut the DNA to leave sticky ends
Palindromic sequences
What is a palindromic base sequence
Where the base sequences on either strand of the DNA are the same as each other- just read backwards
Describe how genes are manufactures in a laboratory using the ‘gene machine’
- The desired sequence of nucleotide bases of a gene is determined from the desired protein that we wish to produce.
- The amino sequence of this protein is determined.
- From this, the mRNA codons are looked up and the complementary DNA triplets are worked out.
- The desired sequence of nucleotide bases for the gene is fed into a computer.
- The sequence is checked for biosafety and biosecurity to ensure it meets international standards as well as various ethical requirements.
- The computer designs a series of small, overlapping single strands of nucleotides (called oligonucleotides) which can be assembled into the desired gene.
- The oligonucleotides are then joined together to make a gene.
- This gene doesn’t have introns or other non-coding DNA.
- The gene is replicated using the polymerase chain reaction.
- The polymerase chain reaction also constructs the complementary strand of nucleotides to make the required double stranded gene.
- It then multiplies this gene many times to give numerous copies.
- Using sticky ends, the gene can then be inserted into a bacterial plasmid.
- This then acts as a vector for the gene, allowing it to be stored, cloned or transferred to another organism in the future.
- The genes are checked using standard sequencing techniques and those with errors are rejected.
What is the advantage of using reverse transcriptase to make DNA fragments
mRNA present in the cell is from actively transcribed genes, so there is lots of the mRNA of interest available to make cDNA.
