L12+13 Recombinant Technology and Sequencing Flashcards
Why are restiction enzymes used
To cleave the DNA into manageable sizes
Why cant large chromosomes be used for analysis
They are too fragile
Common properties of restriction enzymes
Binds as dimers
Recognise specific
Some leave overhangs (sticky ends)
Some cut the DNA flush
Describe the separation of DNA by electrophoresis
DNA loaded into the well at the (negative electode)
Current is applied and DNA moves to the positive electrode (because it has a highly negative charge)
The movement of smaller DNA frgaments is less impeeded so they are able to move further faster
How is DNA then purfied once it has been run by electrophoresis
Use a dye like ethidium bromide to stain the DNA
Small slice of the gel is then taken under UV light
What is the function of ligase
An enzyme which is able to joint two DNA fragements allowing the formation of recombinant DNA
What are cohesive temrinin … Why are the able to ligate
Sticky ends, they are able to hybridise
What are the features of a plasmid
Antibiotic resistance gene e.g. tetracyclin
Origin of replication (allows 50 copies to be made per bacteria
Insert
Characteristics of a plasmid
Small
Extrachromosomal
Circular
Naturally occur in bacteria
Describe how plasmid vectors are usually made
Made from plasmids usually by adding many restriciton sites in one part of the plasmid (multiple cloning site)
How many bases can a plasmid hold (insert)
How many bases bacteria artificial chr
How many bases yeast artificial chr
30K
300K
3 MEGA bases
What two processes does DNA cloning involve
Transformation then selection
What are competent bacteria
How are bacteria engineered to become competent
Bacteria that are ready to take up new DNA
Done by creating temporary holes in the membrane by electroporation or chemical treatment
How efficient is the transformation
How are transformed bacteria selected
Very inefficient
Selected on antibiotic plates
What are the two ways which the starting DNA can be obtained
Genomic libraries
Making a library of cDNA clones
What are the advantages of making a genomic library
What does a genomic library contain
Contains everything
Contains all of the regulatory sequences so transcritpion regulation can be studied
What does a cDNA library contain
Contains only the genes that are active in that cell - only ones where mRNA is expressed
What is the advantage of cDNA libraries
One approach to studying disease - able to compare gene expression in healthy and diseased cells
Describe the process for cloning cDNA
Extract the mRNA from the cell then purify –> reverse transcroptase to form the cDNA which is then ligated into a plasmid and bacteria transformed - these are then bred and purified
What does the cDNA seen in a cDNA library represent
Transcriptome of the tissue
What is important in a cDNA library with regards to isolation of single clones from a mixed population
One insert per plasmid
One plasmid in each bacteria
One bacteria starting each colony
What two classes of genes will be found in a cDNA library
House keeping genes
Tissue specific genes
The ends of all the clones in the library are sequenced, what does this give?
Expressed sequence tags
Stages for formation of a genomic library
Purify and digest chromosomal DNA with restriction enzymes
Plasmid take up and transformation
Clone
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
Explain how the Sanger sequencing method has been automated
Rather than radioactively labelling primers, the ddNTPs used in sequencing are tagged with fluorescent dyes, eye base with a different colour. Then the dsDNA template strand is denatured by heating to break the hydrogen bonds between complimentary base pairs. DNA polymerase and a batch of primers are then added to the mixture and DNA synthesis can occur. At random points in the newly synthesised DNA strand a ddNTP will be incorporated into the strand resulting in termination of DNA synthesis. This will produce DNA strands with ddNTPs at every position in the sequence. These strands can then be separated on a gel allowing the sequences to flow continuously. A camera then measures the fluorescence at a fixed point in the gel as the DNA sequences flow underneath. This camera records the colour of the fluorescence corresponding to each base at each position in the sequence. The results are then presented as a graph showing the intensity of fluorescence over time, this is known as a trace and allows the computer to determine the DNA sequence base-by-base.
What is the main limitation of automated sequencing
Automated sequencing is restricted to 1000-1500 base pairs at a time
Progressive sequencing is one method of sequences genome sequences greater than 1kbps, explain how this process works
You start with a genomic library and genomic sequences. Automated sequencing is then used to determine the 1000bps at either end of the genomic insert, adjacent to the vector sequence. The ends of each clone are sequences using primers that have been designed for the vector sequence. Once the first 1000bps at either end of the genomic insert have been determined, primers are then designed for each of these sequenced regions. Another round of sequencing is then performed to determine the next 1000bp sequence of the genomic insert adjacent to the already determined regions. This process repeats until the sequences produced overlap in the middle.
To sequence large fragments of DNA greater than 1kbp, plasmids are used as a vector, T or F
F – bacterial artificial chromosomes are used
Other than progressive sequencing, what other method has been used to sequence DNA fragments greater than 1kbps and how does this work
Shotgun sequencing. First, a genomic plasmid library is created. The ends clone present in the vector plasmids are then sequenced at random using primers for the vectors. This produces short random sequences from each plasmid that are then assembled by a computer program stitching overlapping regions together to produce a contig. This contig represents the assembled sequence of overlapping regions.
What is are the main advantages of shotgun sequencing
Shotgun sequencing requires no thought and only requires a batch of primers predesigned for the vector sequence rather than needing the design and synthesis of multiple sets of primers. The process can thus be automated
What are the main disadvantages of shotgun sequencing
Because of the random sequences produced it requires the sequencing of more the 6x the size of the genome and is hence very inefficient. It will lead to the sequencing of some regions multiple times and there are always gaps. These gaps require progressive sequencing to be filled in.
Which method was used to sequence the human genome
A combination of shotgun and progressive sequencing
How many base pairs are there approximately in the human genome
3.2x109 – 3bn
How many genes are there in the human genome
23000
When was the human genome started and completed
Started in 1990, finished in 2003
Roughly how long does it take to sequence a human genome now
56 hours
The price of human genome sequencing has decreased from $100m to $8k today, T or F
T
How can gene prediction software be used to identify genes in a sequenced region of DNA
Prediction software can be used to analyse newly sequenced regions of DNA scanning for promoters, start/stop sequences and intron splice sites which would implicate a gene.
What is the problem with gene prediction software
You cannot be sure that predictions are always true and a gene has or hasn’t been identified
How can BLAST software be used to determine gene presence in sequenced DNA
Once the base sequence has been obtained you can use a computer to translate this in all 6 reading frames to derive the corresponding amino acid sequence. BLAST can then be used to search for similarities in the sequence that correspond to similar known proteins
What are the four different ways that the genome can be compared online
Chromosomes can be determined, genes can be predicted, regions of protein homology can be seen and expression data can be provided by comparing expressed sequence tags from a cDNA library
What is meant by high throughput
Small scale, fast and automated
Microarrays allow comparison of the genomes of different tissues, T or F
F – it allows the comparison of the transcriptomes
Explain how microarray grids are made
The grids are made by a precise robot that places a spot on the array for each gene in the genome. Each spot contains one single stranded cDNA antisense strand for the gene. These are attached to a specially treated microscope slide so the ssDNA will stick to it.
Describe how microarrays work
Firstly, the mRNA from a tissue is extracted and purified then tagged with a fluorescent dye. This tagged mRNA is then introduced onto the array and allowed to hybridise to the antisense cDNA in each spot. Only genes being expressed by the tissue will be transcribed and hence present in the mRNA. Thus, these will be the genes to hybridise to their respective cell in the array. A reader then uses a sensitive camera to detect which genes are on/transcribed in the tissue by measuring the fluorescence at each position in the grid. The grid coordinates that exhibit fluorescence can be used to determine the identity of the genes being expressed
Microarrays are an ideal way of comparing gene expression in diseased cells/tissues with the healthy states, T or F
T
What is the disadvantage of reverse mouse genetics
It’s an extremely time consuming and expensive process
How can gene replacement be used to study disease
It enables you to test if a present in human patients causes the disease symptoms in mouse by making the same change in the corresponding mouse gene
How does gene replacement work
Gene replacement is used to make small changes to endogenous genes in mice to see if these elicit diseased phenotypes
Describe the process of creating a construct to knockout a gene in a mice model
First, a genomic clone of the gene is obtained and a selection marker is inserted. The selection marker is usually an antibiotic resistance gene such as NEO which encodes for resistance to neomycin. This selection marker is inserted directly into an exon of the gene hence destroying its action. Outside of the exon another selection marker gene is inserted into the construct, this is known as TK. This creates a construct containing two homologous arms of the target gene that flank the NEO gene and a downstream TK gene.
Once the construct has been designed how is this used to create offspring
The construct is then introduced into mouse embryonic stem (ES) cells using cell culture techniques. The cells DNA repair machinery then recombines the construct into the mouse ES cell genome
What can be said about the recombination of the construct into the mouse genome
The DNA repair mechanism machinery is not very efficient. Either the construct is not inserted into the mouse genome at all, or, non-homologous recombination occurs and the NEO and TK genes are inserted into the mouse genome. The TK gene is inserted into the construct to mark where non-homologous recombination has occurred
How does introduction of the construct into mouse ES cells lead to the production of 3 different cell populations
One population of transformed ES cells will have undergone homologous recombination and contain the knocked-out target gene with the NEO gene in between the homologous arms. Another population that will have undergone non-homologous recombination will have these genes and the downstream TK gene also. The final population will contain ES cells that haven’t integrated the construct at all
How are modified ES cells that contain only the knockout and NEO genes isolated from those that contain also contain the TK gene and those cells that only contain the functional target gene
The cells are cultured in a medium containing neomycin and GANC. The cells that haven’t incorporated the construct will be unable to survive in the antibiotic medium as they don’t contain the resistance gene. Similarly, those cells that have also incorporated the TK gene will be unable to survive also despite having the NEO antibiotic resistance gene. This is because the TK gene makes the GANC medium toxic to them. This will leave only those ES cells containing the construct with the knockout target gene and the NEO gene.
Once the desired ES cells have been obtained that contain the gene knockout, how are mosaic mice produced
The selected cell line containing the knockout gene are reintroduced into mice embryos from a different genetic background to the original ES cells. These embryos are then implanted into back into the female mouse and leads to the production of a first-generation mosaic mouse. These mice contain a mixture of cells from the transformed stem cell line and cells from the mother.
How are mosaic mice then used to produce homozygous knockout mice
Some of the transformed ES cells in the embryo will hopefully have given rise to cells in the gonads. Thus, breeding mosaic animals together will create non-mosaic carries of the transgene in the second-generation. These carriers can then be bred together to produce homozygous mutant animals in the third-generation
Creating homozygous knockout mice can take over a year, T or F
T
What is meant by reverse genetics
Uses a known mutated gene sequence to observe effect on phenotype.
