Sequencing DNA

Question 1

Two contrasting routes of DNA sequencing?

Answer 1

• Molecular Route
• Applications Route

Question 2

What is the molecular route?

Answer 2

This works up from the basics step by step, building on previous knowledge and leading on from the techniques of genetic engineering
and sequencing to their applications and relevance to society.

Question 3

Newer sequencing technologies

Answer 3

involves watching DNA polymerase molecules as they copy DNA - the same
molecules that make new copies of DNA in our cells - with a very fast movie
camera and microscope, and incorporating different colours of bright dyes,
one each for the letters A, T, C and G.

Another entails the use of nanopores to sequence DNA.

Question 4

Sanger sequencing steps?

Answer 4

How Does Sanger Sequencing Work?

Sanger sequencing can be performed manually or, more commonly, in an
automated fashion via sequencing machine.

Each method follows three basic steps, as described below.
1. PCR with fluorescent, chain-terminating ddNTP’s
2. Size separation by capillary gel electrophoresis
3. Laser excitation and detection by sequencing machine

Question 5

Step 1 of sanger

Answer 5

The DNA sequence of interest is used as a template for a special type of PCR called chain-termination
PCR. Chain-termination PCR works just like standard PCR, but with one major difference: the addition
of modified nucleotides (dNTPs) called dideoxyribonucleotides (ddNTPs).

In the extension step of standard PCR, DNA polymerase adds dNTPs to a growing DNA strand by
catalyzing the formation of a phosphodiester bond between the free 3’-OH group of the last nucleotide and
the 5’-phosphate of the next

In chain-termination PCR, the user mixes a low ratio of chain-terminating ddNTPs in with the
normal dNTPs in the PCR reaction. ddNTPs lack the 3’-OH group required for phosphodiester
bond formation; therefore, when DNA polymerase incorporates a ddNTP at random, extension ceases.

The result of chain-termination PCR is millions to billions of oligonucleotide copies of the DNA sequence
of interest, terminated at a random lengths (n) by 5’-ddNTPs.

In manual Sanger sequencing, four PCR reactions are set up, each with only a single type of ddNTP
(ddATP, ddTTP, ddGTP, and ddCTP) mixed in.

In automated Sanger sequencing, all ddNTPs are mixed in a single reaction, and each of the four dNTPs
has a unique fluorescent label.

Question 6

Step 2 of sanger

Answer 6

In manual Sanger sequencing, the oligonucleotides from each of the four PCR reactions are run in four
separate lanes of a gel. This allows the user to know which oligonucleotides correspond to each ddNTP.

In automated Sanger sequencing, all oligonucleotides are run in a single capillary gel electrophoresis
within the sequencing machine.

Question 7

Step 3 of sanger

Answer 7

The last step simply involves reading the gel to determine the sequence of the input DNA. Because DNA polymerase only synthesizes DNA in the 5’ to 3’ direction starting at a provided primer, each terminal ddNTP will correspond to a specific nucleotide in the original sequence (e.g., the shortest
fragment must terminate at the first nucleotide from the 5’ end, the second-shortest fragment must terminate at the second nucleotide from the 5’ end, etc.)

Therefore, by reading the gel bands from smallest to largest, we can determine the 5’ to 3’ sequence of the original DNA strand.

Question 8

What is a DNA Microarray?

Answer 8

The DNA microarray is a tool used to determine whether the DNA from a particular individual contains a mutation in genes like BRCA1 and BRCA2.

On the surface of each chip it contains thousands of short, synthetic, single-stranded DNA sequences,