DNA Sequencing And Analysis

What is DNA sequencing?

The process of determining the precise order of nucleotides within a DNA molecule.

What did Sanger sequencing enable?

Sanger and his team to read sequences of 500-800 bases at a time. The first entire genome that they sequenced as just over 5000 bases long and belonged to a virus that attacks bacteria.

What was the human genome project?

A massive international project in which scientists from a number of countries worked to map the entire human genome, making the data freely available to scientists all over the world

What happens to DNA before it is sequenced?

It is chopped into fragments and each fragment is sequenced.

What do terminator bases do?

Terminator bases stop DNA synthesis when they are included, they contain a fluorescent tag and each base has a different colour.

What is the first step in the capillary method of DNA sequencing?

DNA is mixed with a primer, DNA polymerase and excess of normal nucleotides (with normal bases) and terminator bases.

What happens after DNA is mixed with primer, DNA polymerase and excess DNA nucleotides?

The mixture is placed in a thermal cycler for PCR, which rapidly changes temperature:

• 96°C - double strands split into single strands.

• 50°C - primers anneal to the DNA strand.

• 60°C - DNA polymerase builds up DNA strands by adding nucleotides through complementary base pairing to the template strand.

What happens each time a terminator base is incorporated instead of a normal nucleotide?

The synthesis of DNA is terminated.

Since the terminator bases are present in low amounts and are added at random, what does this mean?

There will be different lengths of DNA fragments depending on where the terminator bases are added. After many cycles, all possible DNA chains will be produced with the reaction stopped at every base.

How are the different DNA fragments separated?

By length, by capillary sequencing, similar to gel electrophoresis in capillary tubes.

How is the final base order able to be identified?

By fluorescent bands, which are detected by lasers, which can allow the order of the sequence to be found (they detect the different colours of the bases).

What does the order of the bases in the capillary tubes show?

The sequence of the new, complimentary strand of DNA which has been made. this allows the sequence of the original strand to the built up. The data is fed into a computer that reassembles the genomes by finding areas of overlap.

One the genome is assembled, what do scientists want to identify?

The genes or parts of the genome that code for specific characteristics. Medical researchers want to identify regions that are linked with particular diseases.

Early on, was working out the base sequence of even short strands of DNA easy?

No, it was difficult and time-consuming using the original Sanger sequencing method.

What have DNA sequencing technologies become?

Faster, and more automated as they have been developed. Recently, technological developments have led to new, automated, high-throughput sequencing processes.

In new techniques, where does the sequencing reaction take place?

On a plastic slide known as a flow cell, rather than using gel or capillaries. Millions of fragments of DNA are attached to the slide and replicated in situ using PCR. The sequencing process still uses the principle of adding coloured terminator bases. All of the clusters are being sequenced and imaged at the same time.