Manipulating Genomes Flashcards
4 steps in genetic engineering
Isolate the gene
Package the gene in a vector ( plasmid)
Transfer the vector into recipient cell
Recipent cell can express the gene
How to isolate a gene
Harvest mRNA from cells where it is being expressed
Use reverse transcriptase to convert mRNA to cDNA
And primers and polymerase to bulid double stranded DNA
Or
Use DNA probe to indetify the gene then use restriction enzymes to cut the gene out
Or
Synthesise from scratch
How is the gene packaged in a vector
Gene is sealed into bacterial plasmids using restriction enzymes
Both dna and plasmid are cut using the same restriction endonuclease
DNA fragments and open plasmid are mixed using DNA ligase
Transfering the vector to the recipent cell
Plasmid must be taken up by the host bacterium by heat shock in the presence of cl- or electroporation
Both a high conc of cl- and electroporation cause the bacterium membrane to become more permable so that plasmid can enter
Process of DNA sequencing
- DNA for sequencing is mized with primer, DNA polymerase and excess of nucleotides and terminator bases
- mixture is placed in a thermal cylinder that rapidly changes temperature at programmed intervals. At 96 degrees the double stranded DNA sequences seperare into single strands, at 50 degrees the primers anneal to the DNA strands
- at 60 degrees DNA polyermase starts to build up new DNA strands by adding nucleotides with complementarybases to the signle strand DNA template
- each time a terminator base is added instead of a normal base the synthesis of DNA stops, this creates DNA strands of different length. These strands are seperated by capillary sequencing
- the order of bases in the cappillary tube shows the sequence of the new, complementary strand of DNA which has been made
Data from sequencing process is fed into a computer which reassembles genomes by comparing fragments that overlap
Applications of DNA profiling
Criminal investigation
Mateeinity and paternity disputes
Immigration cases
Disease analysis- correlatipn between certain patients and increased risk of certian disease, diagnosis of sickle cell anemia
Application of sequencing
Bioinformatics - software tools to anaylse vast quantities of raw biological data
Computational biology - using the data to bulid theorectical models of biological systems
Human genome project - comparisons within species or other species, predisposition to disease, evolutionary history
Analysis of pathogens - sources of disease/ tracking development, analysing genomes for vulnerability / potential treatment
What are restriction enzymes
Enzymes that cleave DNA
They target specifc sequences, recognition sites
May leave sitcky ends, as dna strands are cut in an offset fashion
Bennefit Sticky ends
Sticky ends are complimnetary so base pairs from another strand can anneal
DNA ligase enzyme forms phosphate-sugar backbone to form recombinant DNA
principles of DNA sequences
Aim is to order fragments of dna in size order with terminator bases in order to work out a DNA sequence
Used for criminal investigations and paterinity checks
Process of DNA sequencing
Dna is mixed with primer, DNA polymerase, excess of normal nucleotides and terminator bases
Heated and cooled so DNA strands seperatd and primers anneal
At 60 degrees DNA polymerase starts to bulid up new DNA strands by adding nucletoides
Each time a terminator base is added the synthesis of DNA is terminated resulting in fragments of different length
Fragments are placed into a cappliary tube and terminator bases and therefore DNA code is identified by a computer
Synthetic biology
Designing biological systems from scratch
They include many different techniques:
Genetic engineering
Use of biological systems
Syntheis of new genes to replace faulty ones
Synthesis of an entire new organism
Geniomcs and proteomics
Prediction of amino acid sequcns in a polypeptide
Uses DNA sequence to predict amino acids
What is Bioinformatics
The development of the software and computing tools needed to orgainse and anaylse raw biological data to help us make sense of large quantities of data
What is computational biology
Is the study of biology using computational techniques
Uses the data from bioinformatics to bulid theortical moddls of biological systems which can be used to predict what will happen in different circumstances
