Flashcards in Lecture 14 Deck (34)
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 of each 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
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
The price of human genome sequencing has decreased from $100m to $8k today, T or F
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
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 mutation 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