manipulating genomes

what is recombinant DNA

when fragments of foreign DNA are inserted into other sections of DNA

uses of recombinant DNA

• genetically modified crops - improve their yield by; resisting disease, tolerance to herbicides and pesticides, tolerance to adverse environmental conditions e.g. drought

• genetically modified livestock - can be used to make the production of meat more economically viable; resistant to disease, grow faster and larger 

• increased nutritional value - e.g. rice has been genetically modified to contain vitamin A which is a common deficiency in Asian countries where rice is commonly consumed 

what is a transgenic organism

one that contains DNA from another species within its own

what are palindromic sequences

• sequences of nucleotides

• consist of antiparallel base pairs (pairs that read the same in the opposite direction)

how are DNA fragments produced using restriction enzymes

• DNA is incubated with restriction enzyme

• restriction endonucleases identify the palindromic sequences in the DNA double helix and cut the double-stranded DNA if their recognition sequence is present

• recognition sequences are at either end of the desired DNA fragment and allow restriction enzymes to separate the fragment from the rest of the DNA to obtain the desired gene

• enzymes cut the target gene out via a hydrolysis reaction

• the cut DNA often has sticky ends which can bind to other DNA fragments when they are inserted into vectors

describe the stages in gene transfer

• fragment of DNA is isolated from another organism using restriction endonuclease enzymes

• multiple copies of the gene are made using PCR 

• the gene is inserted into a vector (plasmid or bacteriophage)

• the vector delivers the gene into cells, in a different organism

• cells with the new gene are identified, by using marker genes

• cells with the new gene are cloned

describe the first stage of genetic engineering (inserting DNA fragments into vectors)

• a vector is cut open at a specific site using a restriction enzyme, creating sticky ends

• the same restriction enzyme is used to cut the target DNA fragment, creating complementary sticky ends

• DNA ligase forms phosphodiester bonds between the sugar and phosphate groups on the two strands of DNA, joining the sticky ends of the vector and DNA fragment together

• recombinant DNA is formed 

describe the second stage of genetic engineering (transferring recombinant DNA into host cells)

• involves introducing vectors containing recombinant DNA into host cells, transforming these cells

plasmid vectors;

• small, circular DNA molecules, often found in bacteria

• host cells treated to enhance uptake of plasmids containing recombinant DNA

• e.g. electroporation uses an electrical current to make bacterial membranes more porous, helping plasmids enter bacterial cells

bacteriophages;

• these are viruses that infect bacteria

• they inject their DNA into host bacterial cells during infection

• the phage DNA, now carrying the recombinant DNA, inserts into the host's DNA

describe the third stage of genetic engineering (identifying transformed host cells)

• marker genes identify which cells have taken up the recombinant DNA

• they are inserted into vectors alongside the target genes

• transformed cells are cultivated on selective agar plates

• these transformed cells can be cultured to mass-produce the target DNA fragment 

what is DNA sequencing 

determining the exact sequence of nucleotides in within a DNA molecule 

method of sanger sequencing

• DNA is mixed with primers, DNA polymerase, free nucleotides and a fluorescently labelled modified nucleotide

• DNA is denatured using heat - to make it single-stranded 

• DNA polymerase starts adding free nucleotides to the single-stranded template to start-building new DNA strands 

• once the fluorescently labelled modified nucleotide is added, no more bases can be added (DNA synthesis stops) and each is tagged with a fluorescent colour 

• this produces DNA fragments of all different lengths

• the DNA fragments are separated by length using gel electrophoresis

• a laser detects the fluorescent colours of the DNA fragments to determine their sequence order

• computer software analyses the fragments to reconstruct the original DNA sequence 

how do you sequence a whole genome?

• genome is broken down to even smaller pieces using restriction enzymes

• these sections are places into separate BACs (man-made plasmids) using the enzyme DNA ligase 

• these recombinant BACs are then transformed into E.coli cells which now act as stores for these different sections. 

• each bacteria contains a BAC with a different DNA fragment

• the bacteria divide creating colonies of clones 

• extract the DNA of the BAC from your clone of bacteria (using restriction enzymes) 

• use a restriction endonuclease to digest this DNA. different ones are used to produce different length fragments 

• use electrophoresis to separate the fragments

• each fragment is then sequences in a DNA sequencer machine - this will give you the base code for each fragment

• a computer programme will then analyse overlapping fragments in order to then resemble the whole BAC - giving you the entire genome

why can’t you use the chain termination method to sequence the whole genome 

as the chain termination method only works on fragments up to roughly 750 base pairs long 

what is a BAC

• small pieces of bacterial DNA

• act as a vector to artificially carry DNA into the cell of a bacterium

what is synthetic biology

• where DNA is built from scratch by attaching different genes together 

• therefore the sequence of amino acids in a polypeptide chain can be predicted 

what is bioinformatics 

the development of the software and computing tools needed to organise and analyse data (processing data)

what is computational biology

uses the data from bioinformatics to build theoretical models of biological systems, which can be used to predict what will happen in different circumstances 

what is genetic fingerprinting

used to identify unique DNA patterns in individuals

describe the process of creating a genetic fingerprint 

• DNA extraction - DNA is extracted from a tissue sample and amplified using PCR

• DNA digestion - restriction enzymes are used to cut the DNA into fragments

• fragment separation - gel electrophoresis separates the fragments based on their size, and they are denatured to produce single-strands

• hybridisation - special radioactive probes bind to complementary VNTR (variable number tandem repeats

• development - the positions of the probes are revealed, creating a barcode-like pattern of DNA bands, unique to the individual

what is gel electrophoresis

technique used to separate molecules such as DNA, RNA or proteins based on size by using an electric current applied to an agarose gel matrix 

how do you set up gel electrophoresis

• insert a gel tray with solidified agarose gel into a gel tank

• ensure the wells are close to the negative electrode to position the gel correctly 

• pour a buffer solution over the gel until it is submerged to maintain a constant, suitable PH throughout the experiment