identifying and amplifying Dna Flashcards
discuss 4 different methods of identifying if a DNA sequence is present in a sample and estimating how many of that gene were present; 6 marks
state 2 ways that DNA can be amplified;
1) hybridisation with a radioactive label
DNA is denatured and thenplaced on a nylon/nitrocellulose filter before adding a radioactive complimentary piece of DNA and leaving in conditions to promote hybridisation. excess labels can be washed off before performing autoradiography; if radioactive decay is present then so was the GOI.
shows if the gene is present and how much of it is.
2)Southern blotting
i)cut DNA into smaller pieces using restriction enzymes
ii)perform gel electrophoresis in alkaline solution to cause dsDNA to unwind
iii)press nitrocellulose/nylon filter to the gel to transfer DNA
iiii)bake filter in over to fix DNA to filter
v)add labeled probe and perform autoradiography to check for the presence of the GOI.
shows if the gene is present and how much of it is
3) PCR amplification is an accurate way of estimating how many genes were in the sample.
4) genomic and cDNA libraires
amplificaiton;
1) PCR
2) vectors; bacterial and bacteriohage
what does PCR involve and what are its 6 applications? 12 marks
Polymerase chain reaction
amplification of specific pieces of DNA
1) rare pieces of DNA from complex mixtures can be amplified and then extracted
2)human health applications; amplify an embryos DNA and check for genes that would suggest a specific disease. amplify DNA and check for viral DNA which may suggest disease status
3) the genes can be modified; only the 3’ end of the primer has to be complimentory, the 5’ end can contain a restriction site etc
4)forensices; amplifying DNA from crime scene and performing electrophoresis to create a distinct DNA banding pattern which can be compared to DNA databases
5)paternity testing; perform PCR of child and father to and do gel electrophoresis to check for similar banding
6)agriculture; PCR of embryonic cell to check if the offspring will be female or male (females are preferred)
discuss the 5 components needed for PCR; 5 marks
sample of DNA primers (forward and backward) taq polymerase nucleotide bases Magnesium ions to stabilise the primers
discuss the 3 stages of PCR; 6 marks
1) 95 degrees; denature and unwind dsDNA
2) 55-62 degrees; primers anneal
3) 72; TaqPol extends primers in a 5’ to 3’ direction
how many cycles of PCR are normally done; 1 mark
20-30
what are the 4 ways that PCR can be optimised; 8 marks
correct temperature for annealing; different primers have different optimum annealing temperatures which must be established experimentally; run PCR at different temperatures and check which one gives the highest yield
if temperature is too low then the primers will bind non specifically
if the temperature is too high the primers wont anneal
correct concentration of magnesium; as with annealing temperature there is an optimum which must be established experimentally
correct length of time of each stage of a cycle
correct amount of template and polymerase to begin with; less is more
what are the drawbacks of PCR; 3 marks
TaqPol incorporates errors into the sequence due to no proofreading ability
a small number of contaminating sequences will be produced which contain not only the target sequence but the sequence after that
prior information about the GOI is needed; the end sequence and start sequence of the GOI
how can the total number of copies of the GOI there are
2n (n= number of cycles)
Discuss the use of vectors as a method of DNA amplification; 2 marks
state 3 vectors; 3 marks
discuss in detail 1 of these vectors; 8 marks
a GOI is placed in a vector which is then introduced into a host cell which replicates the vector, looks after the vector (ensures no errors are introduced) and passes the vector down to offspring
1) Bacterial plasmids; size limited
2) Cosmid vectors; larger pieces of DNA can be introduced
3) Yeast artificial chromosomes; larger pieces of DNA can be introduced
bacterial plasmids;
naturally occurring pieces of extra chromosomal circular DNA that normally carry genes beneficial to survival such as toxin production or antibiotic resistance.
plasmids are transferred between bacteria via horizontal transfer.
1)lyse open a cell and isolate a vector from the chromosme. this is easy to do as the two have different properties
2) use a nuclease to open up the vector
3) construct a GOI with complementary sticks ends and combine with vector and ligase enzyme
4)introduce GM vector to a bacteria by applying calcium ions and performing heat shock to make the plasma membrane more permeable. alternatively electroporation can be used
5) becuase the take up efficiency is so low (1 in a million) those which have taken up the vector must be identified by blue/white selection;
bacteria would normally have beta galactosidase protein (omega and alpha peptides) which is capable of breaking X-gal into a blue product. bacteria are modified so they dont have the gene for the alpha peptide. Plasmid has a gene for antibiotic resistance so all those bacteria which grow on plate have the plasmid. plasmid also has the gene for the alpha peptide so beta galactoside can be expressed and the colonies are blue. GOI restriction site is inside the omega peptide gene sequence so all those colonies which are white have the GOI present.
the only problem with using bacterial plasmids to amplify DNA is that the larger the GOI inserted into the vector, the lower the transformation efficiency
state what a bacteriophage is and how they replicate; 5 marks
discuss the use of bacteriophage lambda as a DNA cloning mechanism and as a method of constructing DNA libraries
Bacteriophages are viruses which invade bacreria
They can replicate in two different ways;
1)lysogenic replication; viral DNA inserted into bacterial DNA which is then replicated and passed on to daughter cells
2) lytic replication;viral DNA remain in bacteria but seperate from the bacterial chromosome. it self replicates using bacterial resources; polymerase enzyme moves around the circular piece of DNA continuously and creates a linear piece of DNA which contains many copies of the genome. an endonuclease recognises Cos sites between each genome and cleaves here. each genome can then be inserted into new viral particles which burst out of the cell
The lytic replication pathway can be used to clone DNA by inserting a GOI into the viral DNA
There is alimit to the number of bases that can exist between cos sites, so to increase the size of GOI that can be inserted, the genes relating to the lysogenic pathway are removed (the stuffer fragment)
transformation involving large pieces of DNA is much more efficient when a bacteriophage is used compared to a bacterial plasmid
viral culturing occurs on ‘‘lawns’’ of bacteria; clear spots on the lawn indicate bacterial death and viral lytic replication
discuss both genomic and cDNA libraries
discuss how libraries can be screened and what the libraries can tell you about the organism
genomic libraries are a collection of bacterial colonies, each of which has been infected by a different bacteriophage lambda containing a different fragment of the genome
the genome is first cut into fragments which are then inserted into viral genome in the place where the stuffer region has been removed
complimentory DNA libraries are a collection of bacterial colonies each of which contains different genes from an organism.
The genes are not from the genome but from mRNA which has had reverse transcriptase applied to it, then RNAseH action causes degradation of RNA and then polymerse action to synthesise the complimentary strand
after extracting RNA, the sample is placed on a matrix of repeating T nucleotides so that only the mRNA binds (due to its PolyA tail); the tRNA and rRNA can be washed away
both libraries can be screened to tell you if a particular protein/gene is present. this is much easier than trying to actually screen for one particular protein because this would require you to know a lot about a protein.
a) for example you can lyse the colonies open and apply florescent antibodies and check for binding
b) the agar of the plate may contain a particular nutrient and if some colonies are present this will tell you that the organism contained the enzyme to degrade that nutrient
c) florescent DNA probes can be added and UV can be applied to check for binding
1) genomic libraries tell you about the entire set of introns, exons and promotors present in an organism
2) cDNA libraries tell you what genes were being expressed in the cell taken at that time; gene expression is dynamic temporally and spatially. differential screening of different cells.
3) cDNA tells you about the polyA tails of genes and other mRNA modifications
4) both libraries are needed to map out the intron exon boundires
discuss how DNA is sequenced; 5 marks
what are the limitations to this method; 2 marks
what are solutions to this limitation
Sanger sequencing is still used today
1) mixture is prepared includuing many copies of the ssDNA which is to be sequenced, primers, DNA polymerase enzyme, an abundance of deoxyribonucleotide triphosphates (dNTP’s) and some dideoxyribonucleotides (ddNTP’s) which are florescence tagged
2) DNA replication occurs as normal (dNTP’s added to primer) until a ddNTP is added which terminates the sequence as the polymerase enzyme cant move past the ddNTP
3) the contents of the tube are seperated using gel electrophoresis
4) when the strands reach the end of the gel, a laser and computer detects which florescent tagged ddNTP is present and this can be used to work out the sequence of the DNA
limitations; sequences larger than 1000 bp cant be sequenced. molecules over 1000bp cant have very similar weights so the resolution of gel electrophoresis isint great enough
i)primer walking; different primers are created which bind to different sequences on the DNA of interest, approx 1kb apart. the overlapping sequences are then used to work out the original sequence
ii)shotgun sequencing; a larger piece of DNA is cut into 1kb pieces using restriction enzymes, each of which is cloned in bacterial plasmids before sequencing each one individually
the use bioinformatics software helps scientists make sense of the genomic sequence
next generation sequencing is a term used to describe a series of sequencing techniques which are quicker, easier and cheaper than sanger sequencing; sequencing for example methods which involve sequencing multiple small sequences at once
compare and contrast bacterial plasmids and phage vectors for amplifying DNA
plasmids are inside bacteria and dont require any other organism whereas viral phage vectors requrie bacteria to infect
plasmids are circular whereas phage vector DNA comes in a variety of forms
the transformation efficiency of plasmids is worse than that of phage vectors (10^9 transformants per ug of DNA compared to 10^6)
the efficiency of transformation decreases with size of DNA for plasmids but reamins constant for phage vectors
phage DNA is more difficult to prepare than plasmid DNA
bacterial clones are easier to handle than phage clones
the plating density is limtied (1000 clones per plate) for bacteria but less limited for phages (10,000 phages per plate)
