Recombinant DNA Technology Flashcards
(38 cards)
What is recombinant dna technology?
- General name for taking a piece of dna and combining it with another strand of dna
- Utilises techniques of molecular cloning = amplification, maintenance and manipulation of specific dna
- In vitro and cells to replicate dna
Applications for recombinant dna technology
- Genome organisation
- Production of recombinant proteins (vaccines, therapeutics and enzymes)
- Transgenic organisms
- Gene therapy
What was the first human protein produced by E. coli?
Insulin
Is the genetic code universal?
Yes, understood by all cells, prokaryotic and eukaryotic
Steps of molecular cloning:
- Extraction of relevant nucleic acids
- Determine base sequence of required piece of DNA
- Create many copies of required piece of DNA using PCR
- Clone amplified DNA fragments into a cloning vector to construct recombinant DNA molecules
- Transform recombinant DNA molecules into competent E. coli cells
- Identify recombinant DNA molecules (plasmids) containing the correct DNA sequence
- Application of molecular cloning = eg. gene therapy…
How does molecular cloning initiate?
By amplifying a specific gene by PCR
- The source of DNA can be either nucleic acid extracted from human cells or a DNA library (gDNA or cDNA)
What is the gDNA (genomic dna library)?
A genomic library is a collection of overlapping segments of genomic DNA, cloned into a backbone vector, which statistically includes all regions of the genome of an organism.
What does the gDNA do?
Break up entire genome into small fragments:
- Through restriction endonuclease = enzymes that recognize a specific DNA sequence, called a restriction site, and cleave the DNA within or adjacent to that site
- Mechanical shearing = random fragments
Applications of genomic library:
- Determining complete genomic sequence of an organism
- Source of genomic sequence for generation of transgenic animals through genetic engineering
- Study of regulatory sequences in vitro
- Study of genetic mutations in cancer tissues
What does cDNA responsible of?
A cDNA library is a collection of cloned DNA sequences that are complementary to the mRNA that was extracted from an organism or tissue
Alternative - cDNA
- Cells from tissue are lysed and purified to form mRNA
- mRNA is hybridized with poly-t primer
- Complementary DNA copy is made with reverse transcriptase
- RNA is degraded with RNAse H
- Second cDNA strand is synthesised using DNA polymerase; RNA fragment acts as primer
- Double stranded cDNA copy of original mRNA
Applications of a cDNA library
- Discovery of novel genes
- Cloning of full length cDNA molecules for in vitro study of gene function
- Study of repertoire of mRNAs expressed in different cells or tissues
- Study of alternative splicing in different cells or tissues
An advantage of cDNA over gDNA
It contains uninterrupted coding sequence of gene
- Expression of recombinant protein in bacteria or yeast cell (faster and cheaper method)
- cDNA has no introns
- cDNA library contains the cloned complementary DNA of total mRNA of an organism while the genomic DNA library contains the cloned fragments of the entire genome of an organism.
Which nucleic acid is required?
DNA or mRNA
Which nucleic acid is required for prokaryotes and some lower eukaryotes (yeast)?
DNA
Which nucleic acid is required for higher eukaryotes?
mRNA
How to extract the relevant nucleic acid?
- Cell disruption or cell lysis to expose membrane
- Commonly achieved by chemical or physical method
Remove membrane lipids by adding a….
Detergent
To purify DNA or mRNA from detergents, proteins etc, what is a process is used?
Ethanol precipitation (usually by ice-cold ethanol or isopropanol)
- DNA is insoluble in alcohols - will pellet
- Precipitation improved by increasing of ionic strength e.g addition of sodium acetate
To purify DNA or mRNA from detergents, proteins etc, what is another process is used?
Phenol chloroform extraction
- Phenol denatures proteins - stay in organic phase
- Aqueous phase containing nucleic acid mixed with the chloroform
- DNA isolation - phenol buffered to pH 8
- Also used for RNA isolation - pH 4
To purify DNA or mRNA from detergents, proteins etc, what is the third process that can be used?
Minicolumn purification = expensive but purest dna form
- Nucleic acid bind to solid phase (silica or other)
- Depends on pH and the salt content of the buffer
- Also used for RNA isolation
Method to analyse DNA or RNA
UV absorbance
Abs used to measure the amount of nucleic acid
260nm
Abs used to measure the amount of protein
280nm
