Lecture 13 Flashcards
What is the name given to the sections of DNA produced by restriction enzymes
Restriction fragments
How were restriction enzymes first identified
Restriction enzymes are part of a naturally occurring defence mechanism that digests foreign bacteria
What is the name of the restriction endonuclease that recognises the GAATTC sequence
EcoRI
What attribute of restriction enzymes accounts for their binding to palindromic recognition sites
Restriction enzymes bind as dimers
Restriction enzymes have precise recognition sites, T or F
T
How do restriction enzymes generally cut the DNA
Generally they cut the DNA leading overhangs known as sticky ends
What is meant by blunt restriction enzymes
Restriction enzymes that cut the DNA flush
What process is used to separate restriction fragments once the DNA has been cut
Gel electrophoresis
How are separated DNA restriction fragments visualised after separation
Dyes such as ethidium bromide are added which stains the DNA when exposed to UV light
How are specific fragments then isolated once separated and identified
Specific bands are cut out from the gel using a razor and then the DNA contained within it can be purified out
How is DNA referred to that has been produced by ligation of multiple sequences from different sources
Recombinant DNA
What features of the cohesive/sticky ends allow ligation
The ability of them to hybridise based on complimentary base pairing
In order for sticky end ligation to occur from restriction fragments, the restriction enzymes need to have identical recognition sites, T or F
F – as long as the sticky ends have cohesive overhangs i.e. complimentary bases they can ligate with or without identical recognition sites
DNA cloning involves ligation of DNA fragments into vectors. What vectors are commonly used
Plasmid vectors – small circular, extra-chromosomal DNA that occur naturally in bacteria
What is particularly useful about the vectors used in short sequence DNA cloning
Plasmids have their own very active origin of replication which usually results in 50 copies of the plasmid being make in each bacterium
The vectors used in DNA cloning usually contains antibiotic resistance genes in bacteria and can be transferred onto the progeny and between adult bacterial cells, T or F
T
How are DNA cloning vectors made
Plasmid vectors are made from plasmids by adding a series of restriction enzyme sites in one part of a plasmid called the multiple cloning site
Plasmid vectors can only hold up to 30kbps of DNA, what vector is used for DNA fragments larger than this
Bacterial artificial chromosome (BAC) which can hold up to 300kbps
For fragments between 300kbps and 3Mbps, what vector if best suited
Yeast artificial chromosome
How is transformation of bacteria achieved once recombination of a plasmid vector has occurred
The bacteria are missed with the recombinant plasmid vector and their membranes are permeabilised by electroporation or with chemical treatment. Competent bacteria will take up the new DNA
Transformation of bacteria is an ineffective process, how is this overcome
Integrated into the recombinant plasmid vector is a gene for antibiotic resistance. Once bacteria have been exposed to the vector they are grown on a medium containing that antibiotic. Bacteria that have taken up the plasmid will be the only ones to grow on the medium and thus will contain the target DNA sequence along with the antibiotic resistance gene.
Once the colonies containing the transformed bacteria have grown on the antibiotic medium what is the next stage to produce an unlimited supply of the DNA sequence
Single colonies are lifted from the plate to start a liquid culture. The plasmids can then be easily purified from the bacteria and stored or analysed.
Explain how a genomic library is created
The whole genome of the organism is cut into fragments and each fragment is cloned into a different plasmid vector to create colonies with each fragment sequence
What is the advantage of creating genomic libraries
It contains the entire genome sequence including all genes and regulatory sequences allowing for the study of transcriptional regulation
What is meant by a cDNA library and what is its advantages
cDNA library is created from the mRNA transcribed within a specific cell or tissue. This allows for the study of disease to identify which genes are expressed in a diseases tissue compared to a normal healthy tissue
What is meant by the transcriptome
All genes expressed by a cell or tissue
How are cDNA libraries created
Firstly, mRNA is extracted from a cell. Reverse transcriptase then coverts this single stranded mRNA to dsDNA – referred to as cDNA. The cDNA from each mRNA is then cloned and undergoes ligation and transformation to get each sequence into bacteria.
What three things are important when creating cDNA libraries from mRNA
Only one cDNA insert is inserted into each plasmid, this is done by controlling concentrations. Once plasmid only must be taken up into each bacterium and one bacteria must start each colony.
Housekeeping genes are often cloned more often when creating cDNA libraries, why is this
They are highly transcribed
Describe the process of dideoxy terminator/chain termination/Sanger sequencing
Firstly, you start with a dsDNA sequence of interest and this is denatured by heating to 100?C to break the hydrogen bonds between complimentary base pairs and leave ssDNA templates. The template strand is then allowed to cool in the presence of radioactively labelled primers allowing them to anneal. DNA synthesis is then allowed to occur by adding DNA polymerase and deoxynucleotide trisphosphates. A mix of dideoxy nucleotide trisphosphates are also added into the mix, that prevent the subsequent synthesis of DNA once they have been incorporated into the growing polynucleotide strand. By having a mix of deoxy and dideoxy nucleotides, different strands will end at different positions. These strands can then be separated on a gel to produce distinct bands based on template strands that have terminated at each position. Running all four ddNTP reactions on the same gel results in a nucleotide ladder which allows for sequencing base by base.
What is the difference between normal nucleotides used in DNA synthesis and those used in dideoxy terminator sequencing
Whereas deoxynucleotide trisphosphates contain a 3’ C-OH bond, dideoxy nucleotide trisphosphates have a 3’ C-H bond which DNA polymerase cannot act on
Describe the structure of a primer and how they are made
A primer (oligomer) are short, roughly 20 nucleotide single stranded DNA sequences that are usually synthesised chemically. To synthesise primers, a small part of the DNA sequence of interest must be known. This is usually part of the vector sequence in which the target sequence is immediately integrated into after.
Why is dideoxy terminator sequence no longer used
Requires a lot of time and effort to read off the gel base by base. It also requires the primers to be radioactively labelled and left over night on an X-ray film to allow visualisation of the strands
How has the dideoxy terminator sequencing method been improved
Rather than labelling the primers the ddNTPs are labelled themselves, each base with a different colour
