Give an account of the methods used in sequencing the human genome.
Sequencing of human genome has made it possible to understand the link between various genes and their functions.
If there are any gene defects that express as disorders or that increase the susceptibility of an individual to a disease then specific gene therapies can be worked out
Methodologies of human genome sequencing
The methods involve two major approaches
(i) Expressed Sequence Tags (ESTs) This method focusses on identifying all the genes that are expressed as RNA.
(ii) 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, first the total DNA from cell is i.e., solated and broken down in relatively small sizes as fragments.
There 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 (YACs).
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.
Dr. Alec Jeffreys developed the technique of DNA fingerprinting in an attempt to identify DNA marker for inherited diseases.
DNA fingerprinting uses short nucleotide repeats called Variable Number Tandem Repeats (VNTRs) as markers. VNTRs vary from person to person and are inherited from one generation to the next. Only closely individuals have similar VNTRs.
Replication was allowed to take place in the presence of radioactive deoxynucleotides precursors in E.coli that was a mutant for DNA ligase. Newly synthesised radioactive DNA was purified and strands were separated by denaturation. These were centrifuged using density gradient centrifugation. Which of the following would be a correct result?
In above case, as E.coli is a mutant for DNA ligase, it will result in no further joining of Okazaki fragments on lagging strand.
This will ultimately result into the formation of both high molecular weight fragments (on leading strands) and low molecular weight fragments (on lagging strand). Hence, only the graph (a) could be the appropriate result after centrifugation.