Recombinant DNA technology Flashcards
Polymerase Chain reaction (PCR)
Identifies and amplifies rare DNA sequences directly out of complex DNA mixtures providing you have some sequence information
- catalysed by DNA polymerase and initiated by short olgionucleotide primer which matches end of the template DNA.
- denaturation of strands- at 95degrees, separates the strands to allow the two complimentary primers to bind.
- annealing of primer- at 60 degrees, it is similar to the melting temperature of the primer- template DNA sequence, hence allows accurate binding of the primers
- extension - occurs in the 5’ to 3’ and occurs at 72 degrees as primer binding will not occur at this temp, but synthesis can
Ingredients for PCR
DNA template- provides the starting material and contains the sequence which is desirable for amplification
Primers- base pair to the ends of the desired sequence and are necessary for initiation of synthesis
Nucleotides- a pool of free dNTPs are sequentially added by DNA polymerase to the growing chain of new DNA
Taq polymerase- a special heat-tolerant polymerase that catalysed the synthesis reaction
Suitable buffer- (pH 7) is always necessary for biological reactions, and the addition of magnesium as it is a co-factor for DNA polymerase
Gene cloning
Bacteriophages and Plasmid DNAs used to transfer DNA into other cells to isolate, multiply or express the insert into the target cell.
Usually consists of
-Insert (gene of interest)
-Backbone of the vector (necessary for replication and selection)
-ORI- origin of replication (in a host cell)
-Selectable markers- genes which allow you to tell if the plasmid is present in the host cell
-Multiple cloning site used to insert your gene of interest using restriction enzymes
- a promoter before the gene which may or may not be the same as the genes original promoter
Recombinant protein production
Recombinant DNA is DNA that is artificially created from two or more sources and is incorporated into a single recombinant molecule. Eg:
- recombinant factor VIII is the antihaemophilic factor A- haemophilia caused by a deficiency in this. Hence, making this recombinant molecule has significantly reduced this disease.
Which expression system to use
Bacteria are easy to manipulate and they grow and divide quickly. However some eukaryotic proteins cannot be produced in an active form in E. Coli cells. This may be due to codon usage, lack of intron splicing, cannot carry out post translational modifications. Therefore need to use eukaryotic system to solve problem, including yeast cells, insect cells and mammalian cells.
Cloning DNA
- Find a cloning vector
- Precise cutting of DNA- restriction endonucleases
- Joining fragments of DNA - DNA Ligase
- Increasing the amount of DNA available - recombinant DNA, cloning vectors: plasmid, bacteriophage)- polymerase chain reaction
- Identifying host cells with recombinant DNA- selection
Libraries
Collection of DNA sequences in cloning vectors
- each plasmid has a different DNA sequence inserted and can be individually clones
- genomic DNA library- fragments of genomic DNA cloned into vector
- cDNA library- cDNA derived from mRNA cloned into vector, cDNA library represents genes being expressed.
Gene expression - cDNA
RNA sequences are amplified by conversion to DNA
mRNA template- decreased reverse transcriptase- cDNA
cDNA can be clones- Gene sequences as expressed (no introns, poly-A tail)
Foreign genes in plasmids can be expressed as protein.
Screening a cDNA library
Now have mixture of cDNA molecules in E. Coil cells, next comes task of isolating cDNA encoding specific protein of interest. Ie screening the library.
Success depends on: strategy adopted for screening, abundance of mRNA, luck
Many methods available for screening:
- nucleic acid hybridisation
- antibody screening
- biological activity
Micro arrays
Analyse the expression of thousands of genes at the same time- multiple reactions on a single chip which allows automation of analysis
A DNA microarray has individual spots for thousands of individual DNA fragments representing separate genes. Each sequence matches a specific position on the grid.
Mutagenesis
Can be:
- site directed: specific nucleotide/s mutated to obtain desired sequence using recombinant DNA technology.
➖incorporation of specific nucleotides into primers
➖ can be insertion, deletion, point mutation, frame shift etc
➖parent molecule is converted to the mutated version by PCR using mutated primers
- random: eg ENU- useful for classic genetic studies. Screen mutants to find desired characteristic.
DNA sequencing
Taq polymerase has BP error of 1/1000. Need to check our DNA is correct through sequencing.
- most common method is Sanger dideoxy method or chain termination method
- Develop a primer upstream to sequence of dna that you want to know. If it is a gene, you can make a primer against the promoter section which is generally conserved. Alternatively, if it’s a vector, you can make the primer against a known part of the vector upstream of the MCS (only need one primer as you can deduce the complimentary sequence)
- determines exact nucleotide sequence of any cloned DNA
- methods synthesises DNA from the DNA fragment of interest. Results in production of a series of daughter strands that are labelled at one end and differ in length by one nucleotide
- daughter strands separated by gel electrophoresis
- fluorescent labels identify each base- all truncated fragments that fluoresce one colour eg. G- black
Ingredients for sequencing
- 4 tubes containing: Template DNA Primer DNA polymerase All 4 dNTP's One modified nucleotide Each tube contains different modified nucleotide
Dideoxynucleotide (ddNTP): an artificial molecule that lacks a hydroxyl group at both the 2’nand 3’ carbons of the sugar moiety.
Deoxynucleotide triphosphate dNTP: contains hydroxyl group on 3’ sugar- allows formation of phosphodiester bond between two nucleotides
Four separate reactions in gene sequencing
- as reaction proceed, nucleotides are incorporated into the growing strand. The concn of modified nucleotide is 100x less than the normal nucleotides, mostly, the normal nucleotides will be incorporated. Every now and then, the modified nucleotide will be incorporated into growing strand. In first tube, this will be at G, second tube at C, third tube at A and last tube at T
- whenever this happens, the growing strand will terminate, as no new nucleotide can be added if there is no 3’OH on the previous nucleotide
- since the concentration of the modified nucleotide is low, the growing strand won’t terminate at the first base, but over time, will terminate at different locations along the strand.
- the result is, for example in the G tube, a mixture of varying lengths of growing DNA all finishing at G locations in the DNA
Analysis of gene sequencing
Since no further nucleotides are going to be added once the ddNTP has been incorporated, a dye molecule can be linked to the ddNTPs at the start. If this happens, the mix of stands are all dye-labelled. One benefit of this is that not radioactivity needs to be involved, but more importantly, each ddNTP can be linked to a different coloured dye. The four mixtures are all then mixed together and run down a thin gel.
A detector can then move along the gel and detect the colour of each band, giving a chromatograph of the sequence.
