Lecture 14

Question 1

Explain how the Sanger sequencing method has been automated

Answer 1

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.

Question 2

What is the main limitation of automated sequencing

Answer 2

Automated sequencing is restricted to 1000-1500 base pairs at a time

Question 3

Progressive sequencing is one method of sequences genome sequences greater than 1kbps, explain how this process works

Answer 3

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.

Question 4

To sequence large fragments of DNA greater than 1kbp, plasmids are used as a vector, T or F

Answer 4

F – bacterial artificial chromosomes are used

Question 5

Other than progressive sequencing, what other method has been used to sequence DNA fragments greater than 1kbps and how does this work

Answer 5

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.

Question 6

What is are the main advantages of shotgun sequencing

Answer 6

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

Question 7

What are the main disadvantages of shotgun sequencing

Answer 7

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.

Question 8

Which method was used to sequence the human genome

Answer 8

A combination of shotgun and progressive sequencing

Question 9

How many base pairs are there approximately in the human genome

Answer 9

3.2x109 – 3bn

Question 10

How many genes are there in the human genome

Answer 10

23000

Question 11

When was the human genome started and completed

Answer 11

Started in 1990, finished in 2003

Question 12

Roughly how long does it take to sequence a human genome now

Answer 12

56 hours

Question 13

The price of human genome sequencing has decreased from $100m to $8k today, T or F

Answer 13

T

Question 14

How can gene prediction software be used to identify genes in a sequenced region of DNA

Answer 14

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.

Question 15

What is the problem with gene prediction software

Answer 15

You cannot be sure that predictions are always true and a gene has or hasn’t been identified

Question 16

How can BLAST software be used to determine gene presence in sequenced DNA

Answer 16

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

Question 17

What are the four different ways that the genome can be compared online

Answer 17

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

Question 18

What is meant by high throughput

Answer 18

Small scale, fast and automated

Question 19

Microarrays allow comparison of the genomes of different tissues, T or F

Answer 19

F – it allows the comparison of the transcriptomes

Question 20

Explain how microarray grids are made

Answer 20

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.

Question 21

Describe how microarrays work

Answer 21

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

Question 22

Microarrays are an ideal way of comparing gene expression in diseased cells/tissues with the healthy states, T or F

Answer 22

T

Question 23

What is the disadvantage of reverse mouse genetics

Answer 23

It’s an extremely time consuming and expensive process

Question 24

How can gene replacement be used to study disease

Answer 24

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

Question 25

How does gene replacement work

Answer 25

Gene replacement is used to make small changes to endogenous genes in mice to see if these elicit diseased phenotypes

Question 26

Describe the process of creating a construct to knockout a gene in a mice model

Answer 26

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.

Question 27

Once the construct has been designed how is this used to create offspring

Answer 27

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

Question 28

What can be said about the recombination of the construct into the mouse genome

Answer 28

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

Question 29

How does introduction of the construct into mouse ES cells lead to the production of 3 different cell populations

Answer 29

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

Question 30

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

Answer 30

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.

Question 31

Once the desired ES cells have been obtained that contain the gene knockout, how are mosaic mice produced

Answer 31

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.

Question 32

How are mosaic mice then used to produce homozygous knockout mice

Answer 32

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

Question 33

Creating homozygous knockout mice can take over a year, T or F

Answer 33

T

Question 34

What is meant by reverse genetics

Answer 34

Uses a known mutated gene sequence to observe effect on phenotype.