3.8.4.1 Recombinant DNA Technology – AQA A Level Biology Summary Notes
Recombinant DNA
- DNA from two or more sources that have been combined together
- Transfer of fragments of DNA from one organism, or species, to another
+ allows humans to directly genetically modified organisms by inserting DNA into their genome
The genetic code is UNIVERSAL, as are transcription and translation mechanisms, so transferred DNA
can be translated within cells of the recipient (transgenic) organism
Transgenic organism (genetically modified)
- Organism that contains recombinant DNA
How do we create a transgenic organism? (Briefly, then will outline below in more detail for each
step)
1. Isolation of desired DNA fragment (likely to be a gene)
2. Insertion of DNA fragment into a vector to form recombinant DNA
3. Transformation – transfer of recombinant DNA into host cell
4. Identification of the host cells that have been successfully transformed
5. Growth/cloning of successfully transformed host cells, and potentially harvest protein
Fragments of DNA can be produced by several methods
- Conversion of mRNA to complementary DNA (cDNA) using reverse transcriptase
- Using restriction enzymes to cut fragment containing desired gene from DNA
- Creating the gene in a ‘gene machine’
Conversion of mRNA to complementary DNA (cDNA) using reverse transcriptase
- first possible method uses enzyme reverse transcriptase
- Example: if we want copies of human insulin gene to place into bacterium and we have
access to human pancreatic cells in lab, these cells will contain lots of copies of mRNA coding
for insulin
- Cells can be lysed (split open), then the enzyme reverse transcriptase and free DNA
nucleotides added
- DNA nucleotides will complementary base pair to mRNA template, then reverse
transcriptase will join DNA nucleotides together, catalysing phosphodiester bonds, to form
complementary DNA
, - We have created a RNA/DNA hybrid; the RNA strand is hydrolysed and DNA polymerase is
used to replace RNA strand with DNA
Two advantages of obtaining DNA fragments using reverse transcriptase
1. mRNA is present in large amounts in cells that make large quantities of specific proteins,
therefore easy to obtain GoI
2. mRNA does not contain introns, due to splicing having occurred
Using restriction enzymes to cut fragment containing desired gene from DNA
- second method
Restriction endonuceleoases
- cut phosphodiester bonds of DNA at specific palindromic base sequences called restriction
sites
- Some cut DNA symmetrically leaving blunt ends
- Others cut DNA asymmetrically leaving ‘sticky ends’, which are useful later on in genetic
engineering process
If restriction sites are found in a chromosome, flanking GoI is obtained through this method
Creating the gene in a ‘gene machine’
- third method (artificial method)
Creates a gene with a base sequence of your choice using free DNA nucleotides and DNA polymerase
Recombinant DNA
- DNA from two or more sources that have been combined together
- Transfer of fragments of DNA from one organism, or species, to another
+ allows humans to directly genetically modified organisms by inserting DNA into their genome
The genetic code is UNIVERSAL, as are transcription and translation mechanisms, so transferred DNA
can be translated within cells of the recipient (transgenic) organism
Transgenic organism (genetically modified)
- Organism that contains recombinant DNA
How do we create a transgenic organism? (Briefly, then will outline below in more detail for each
step)
1. Isolation of desired DNA fragment (likely to be a gene)
2. Insertion of DNA fragment into a vector to form recombinant DNA
3. Transformation – transfer of recombinant DNA into host cell
4. Identification of the host cells that have been successfully transformed
5. Growth/cloning of successfully transformed host cells, and potentially harvest protein
Fragments of DNA can be produced by several methods
- Conversion of mRNA to complementary DNA (cDNA) using reverse transcriptase
- Using restriction enzymes to cut fragment containing desired gene from DNA
- Creating the gene in a ‘gene machine’
Conversion of mRNA to complementary DNA (cDNA) using reverse transcriptase
- first possible method uses enzyme reverse transcriptase
- Example: if we want copies of human insulin gene to place into bacterium and we have
access to human pancreatic cells in lab, these cells will contain lots of copies of mRNA coding
for insulin
- Cells can be lysed (split open), then the enzyme reverse transcriptase and free DNA
nucleotides added
- DNA nucleotides will complementary base pair to mRNA template, then reverse
transcriptase will join DNA nucleotides together, catalysing phosphodiester bonds, to form
complementary DNA
, - We have created a RNA/DNA hybrid; the RNA strand is hydrolysed and DNA polymerase is
used to replace RNA strand with DNA
Two advantages of obtaining DNA fragments using reverse transcriptase
1. mRNA is present in large amounts in cells that make large quantities of specific proteins,
therefore easy to obtain GoI
2. mRNA does not contain introns, due to splicing having occurred
Using restriction enzymes to cut fragment containing desired gene from DNA
- second method
Restriction endonuceleoases
- cut phosphodiester bonds of DNA at specific palindromic base sequences called restriction
sites
- Some cut DNA symmetrically leaving blunt ends
- Others cut DNA asymmetrically leaving ‘sticky ends’, which are useful later on in genetic
engineering process
If restriction sites are found in a chromosome, flanking GoI is obtained through this method
Creating the gene in a ‘gene machine’
- third method (artificial method)
Creates a gene with a base sequence of your choice using free DNA nucleotides and DNA polymerase
