what are the steps in sanger sequencing?
1) create copies of the sequence
- heat to separate the strands
- cut into fragments
- create copies
2) create complementary strands to fragments
- mixture of DNA fragments and: nucleotides, terminating nucleotides, primers, DNA polymerase
- polymerase uses primers to attach to fragments
- nucleotides added until a terminator is added as terminator cant form phosphodiester bonds as it lacks and OH group
3) analyse complementary fragments
- separate with gel electrophoresis
which method is better than sanger sequencing and why?
high-throughput
- cheaper
- automated
- rapid
what are the benefits of DNA sequencing?
- genome-wide comparisons between individuals and species to reveal how related they are
- predict amino acid sequence of genes to reveal tertiary structure of proteins
- useful for synthetic biology to develop new drugs
what three molecules does gel electrophoresis separate?
- DNA
- RNA
- proteins
how does gel electrophoresis work?
- put a line of wells in agar gel
- submerse in buffer solution
- place a -ve electrode at the end with the wells and a +ve electrode at the opposite end
- the lighter the DNA segment the closer to the +ve electrode it will be
what is gel electrophoresis used for?
- genome sequencing
- DNA profiling
what is genetic engineering?
isolating a gene from one organism and placing into another organism
why does genetic engineering work?
genetic code is universal
what are the uses of genetic engineering?
- gene therapy
- modify plants
- modify pathogens (carry medicines)
- pharming
what is pharming?
- animal DNA is altered to produce human proteins for medicine
- testing drugs
what does PCR do?
rapidly produce millions of copies of DNA segments
what is the PCR method?
- requires a thermal cycler and primers
- 95 degrees to separate DNA strands by breaking hydrogen bonds
- 55 degrees for primers to bind to the ends of the strand
- 75 degrees so TAQ polymerase can add bases to the strands so there are 2 of the starting DNA strand
what are the three main steps in pcr?
- denaturing
- annealing
- synthesis
how does the enzyme structure of an extremophile differ to regular enzymes?
more disulphide bridges on the inside away from the heat
why is TAQ polymerase used in pcr?
- obtained from bacteria in hydrothermal vents so can withstand high temps without denaturing
- PCR can be cycled repeatedly without stopping to replace enzymes
how do we isolate the desired gene in genetic engineering?
- restriction endonuclease recognises specific sequences in DNA and cuts strand
- covalent, phosphodiester and hydrogen bonds are hydrolysed
- sticky ends form on the gene
what are sticky ends?
ends of a gene that have the ability to form hydrogen bonds with complementary bases
how is recombinant DNA formed in genetic engineering?
- a vector (usually a plasmid) is cut with same restriction endonuclease as the gene and leaves more sticky ends
- DNA ligase is used to reform phosphodiester bonds
how is modified plasmid inserted into host in genetic engineering?
- electroporation which temporarily disrupts cell wall/membrane
- microprojectile gene gun
- Ca2+ and heat which temporarily disrupts cell wall
- liposomes which enter cell by endocytosis
what is replica plating?
- transfer technique that may be used to produce multiple copies of a particular colonial arrangement on a number of agar plates
- with this technique, cells from bacterial colonies can be transferred from one plate (a master copy or template) to several new plates, and the relative arrangement of the colonies remains fixed
what are the advantages of genetic engineering?
- prevent and fight disease
- vaccines
- gene therapy
- reproductive technologies
what are the disadvantages to genetic engineering?
- long terms effects not known
- embryo use could be classed as murder
- animal exploitation
- costly and time consuming
what is xenotransplantation?
the process of grafting or transplanting organs or tissues between members of different species
