Question
Which methodology is used while sequencing the total DNA from a cell? Explain it in detail.
✓
Answer
Methodologies of HGP:The methods involve two major approaches:
- Expressed sequence tags (ESTs): This method focusses on identifying all the genes that are expressed as RNA.
- Sequence annotation: It is an approach of simply sequencing the whole set of genome that contains all the coding and non-coding sequences, and later assigning different regions in the sequence with functions.
- For sequencing, the total DNA from cell is first isolated and broken down in relatively small sizes as fragments.
- These DNA fragments are cloned in suitable host using suitable vectors. When bacteria is used as vector, they are called bacterial artificial chromosomes (BAC) and when yeast is used as vector, they are called yeast artificial chromosomes (YAC).
- Frederick Sanger developed a principle according to which the fragments of DNA are sequenced by automated DNA sequences.
- On the basis of overlapping regions on DNA fragments, these sequences are arranged accordingly.
- For alignment of these sequences, specialised computer-based programmes were developed.
- Finally, the genetic and physical maps of the genome were constructed by collecting information about certain repetitive DNA sequences and DNA polymorphism, based on endonuclease recognition sites.
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