BIOL-3080 1: DNA Sequencing

Why have we sequenced the genomes of animals like mosquitos and bees?

Why have we sequenced the genomes of animals like mosquitos and bees?

Because they have medical and economic importance.

Why does commercial DNA analysis use genotyping rather than whole genome sequencing?

Genotyping detects SNPs associated with genetic conditions, diseases, rare genes. Genotyping is cheaper.

What is the shotgun strategy of genome sequencing?

Small fragments of DNA are sequenced in no particular order and re-assembled “in silico” to reconstruct the original order of the sequence in genomic DNA.

What does “in-silico” mean?

Read on a computer.

What does a gene library consist of?

The collection of vector DNA isolated from transformed bacterial cells.

What is a contig?

A series of overlapping DNA sequences used to make a physical map that reconstructs the original DNA sequence.

In an ideal scenario, what does the longest contig represent?

The length of the genome.

What is the primer walking strategy of genome sequencing?

Start sequencing from a specific site in genomic DNA, design a primer at a site based on the sequence obtained, start sequencing with the new primer and repeat.

What is the shotgun strategy used for?

Sequence small regions of DNA or close gaps in contigs.

What are two disadvantages to the primer walking strategy?

It requires new primers for each extension, making the process more expensive and time-consuming.

What DNA sequencing strategy was used to obtain the first full genome?

Sanger’s method

What is the general definition of Sanger’s method?

The in vitro synthesis of DNA in the presence of modified nucleotides (ddNTP).

What does the incorporation of a ddNTP during DNA synthesis result in?

Chain termination.

Where is the radio label located on DNA molecules produced by the Sanger method?

on the 5’ ends

What would happen if too much ddA is present in a sample?

All resulting DNA fragments are very short.

How many reactions are needed in the Sanger method?

4 reactions, each containing a different ddNTP (ddA, ddT, ddC, and ddG).

How is the original sequence determined from the 4 reactions used in Sanger’s method?

The reactions loaded into an agarose gel and the bases are read by counting bands from bottom to top.

What is the difference between the gel used in Sanger’s method and those used in our labs?

It is an extremely high resolution gel capable of separating fragments with a single nucleotide difference.

What is the process of reading radio-tagged DNA fragments in an agarose gel called (Sanger’s method)?

Autoradiography.

Where is the shortest DNA strand found in gel electrophoresis?

At the bottom of the gel.

What is the compression phenomenon?

The compression of bands at the top of a gel prevent an accurate reading of the band order.

What is the cause of bands appearing at the same position in each nucleotide lane of a Sanger method gel?

A structural problem in the DNA (example: hairpin structure).

What is another term for hairpin structures?

Homopolymeric DNA.

How do we get around the problem of not knowing what primer to design when there is no previous sequence information known about the DNA template?

Use standard vector primers.

How many primers are annealed from the standard vector plasmid?

Only one primer is annealed from the denatured vector.

What would happen if you anneal the forward and reverse primers at the same time in a single sample?

You would obtain 2 opposing sequences that would conversely overlap each other.

Why is it common to obtain the sequence of both strands of the same double-stranded DNA in two separate samples?

You can check for complementarity and identify errors.

What are the major steps required to obtain a “reference genome”?

DNA extraction —> DNA fragmentation —> Clone into vectors —> Transform bacteria, grow, isolate vector DNA —> Sequence the library —> Assemble contiguous fragments.

What are some problems with manual DNA sequencing?

• Can read only 150-200 nucleotides per gel

• Too labour intensive and time-consuming

What is dye-terminator sequencing?

Uses a different colour fluorescent dye to tag each ddNTP, producing coloured signals read on a chromatograph.

What does it mean that dye-terminator sequencing is multiplex?

All 4 reactions can take place in the same tube and be loaded into the same lane on a gel.

What are the advantages of automated DNA sequencing?

• Can read up to 900 nucleotides per reaction

• Allows automated reading and recording of results

• Cost effective

• Multiplexing

• Can sequence 384 different DNA samples simultaneously using capillary gels

What is the disadvantage of Second Generation DNA sequencing over the Sanger method?

It is geared toward a large number of samples and requires high computing and data storage capacities.

What major steps in genome sequencing can be eliminated by Next Gen sequencing?

• Inserting/cloning the DNA into a vector

• Transformation of vector into bacteria

• Isolation of plasmid or bacterial DNA from transformed bacteria

After DNA fragmentation, what are the added steps in Next Gen sequencing?

• Ligate “adapter sequences” to each end of the DNA sequence

• PCR amplification

How are the primers in Next Gen sequencing designed?

They complement the known sequences of the adapter tags

Why is PCR amplification needed in Next Gen sequencing?

No bacteria was used to amplify the target sequence.

What is Illumina sequencing?

DNA sequencing by synthesis that does not involve permanent chain termination.

What is the multiplexing ability of Illumina sequencing?

Up to 50 million spots can be analyzed at the same time.

What is Nanopore squencing?

A Third Gen sequencing method that monitors changes to an electrical current as single-stranded DNA moves through a tiny pore in a membrane.

What is the advantage of Nanopore sequencing?

It can be done in the field using a small portable device (MinION).

What are the disadvantages of PacBio (SMRT) sequencing over the Illumina method?

• Higher error rate

• Increased cost

• Not a commonly accessible technology

What is single molecule real-time sequencing (SMRT)?

The uninterrupted DNA synthesis performed by a single DNA Polymerase, observed in real time.

Where is the fluorescent tag attached to the dNTP in SMRT?

The gamma phosphate.

What are major steps in SMRT?

A single DNA polymerase is attached to the bottom of a microwell —> As the labeled dNTP is held by DNA pol, the laser detector records a pulse of coloured light —> When the dNTP is incorporated, the gamma phosphate is cleaved off and the light is lost —> The next dNTP added by DNA pol will give off another pulse of coloured light.

What do the differences in height and width of peaks obtained in SMRT signify?

Nothing, they are completely random.

What are the three examples of large scale genome sequencing projects given in lecture?

• Personal Genome Project

• The Cancer Genome Project

• Pediatric Cancer Genome Project

How many reading frames are there in double-stranded DNA and what are they?

There are 6 total frames: 1, 2, 3, -1, -2, and -3

How is the correct reading frame usually determined?

The reading frame with the longest “open” DNA sequence (no stop codon encoded).

How long is the smallest naturally-occurring protein?

~100 amino acids.

What is an open reading frame?

The longest sequence of DNA that encodes a continuous stretch of amino acids before encountering a stop codon.