Question

1 ) why does Sanger sequencing need large amount of input material?

2) Two features that distinguish HTS (high throughput sequencing ) from Sanger are

a) HTS requires a small amount of input DNA

b)No information about the input DNA sequence is required in HTS

Describe how HTS achieves  these features

Answer 1

1) DNA sequencing includes several methods and technologies that are used to determine the order of the nucleotide bases- Adenine, guanine, cytosine and thymine- in a molecule of DNA.

Sanger sequencing also known as chain termination method involves use of dideoxynucleotide triphosphate as DNA chain terminators.

The classical chain termination method requires 1. a single stranded DNA template, 2. a DNA primer, 3. a DNA polymerase, 4. dideoxynucleotide triphosphate and modified nucleotides that terminate DNA strand elongation. In addition to this, gel electrophoresis is also needed to observe the bands of DNA.

To achieve chain elongation and then subsequentely termination all the ingredients/materials listed above are required, missing one of them will not give the data of sequencing and thereby the correct base present in the DNA strand .

2) In case of High Throughput Screening (HTS), sequencing of DNA templates does not require the physical separation of DNA template involved in Sanger sequencing.

Example - Pyrosequencing uses sequencing by synthesis as the sequence read out can be achieved at the same time as the sequence is extended. Therefore, electrophoresis, as used in Sanger sequencing is not needed.

Please try to read methods of Sanger sequencing and HTS in detail. It will help to understand the answer in a better way.