How could you perform a complete sequence of a genome? What experiments would be performed? How...

How could you perform a complete sequence of a genome? What experiments would be performed? How can you create an entire database with the complete genome sequence?

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Expert Solution

Genome sequencing or DNA sequencing contains biochemical methods for determining the order of nucleotide bases in DNA molecule. It include many techniques like Chain termination, pyrosequencing and chain degradation methods, etc.

Chain termination (sangers method): This method depends on ddNTP i.e dideoxyribonucleoside triphosphate. which lack 3'OH group. It contain short single stranded DNA molecule which will be sequenced using DNA polymerase, short DNA primer to start polymerization, and deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, dTTP) in the reaction mixture if a dideoxy analog of any of these nucleotide is present it will stop the chain progression as it lack 3'OH and cannot form phosphodiester bond. To determine sequence of DNA fragments, they are separated in single strands and sequenced using ddNTPs in four different reaction mixture having different ddNTPs in each reaction. The products are separated by PAGE electorphoresis in parallel lane. in AUTOMATED SEQUENCING same technques is used just dideoxy are flouroscently labeled with different tags of different color. As the sequence terminates floroscence is seen.

Chemical degradation: sequenced by Maxam-Gilbert. Double stranded DNA is firstly labeled floroscently at 5' end and separated into sinle strand by denturating electrophoresis. four aliquot are taken and treated with different mixtures. each molecule get modified at one position and then samples are separated by breaking phosphodiester bonds between modified molecules. these cleaved molecules are separated by polyacrylamide electrophoresis and sequenced similiar to chain termination method.

Pyrosequencing: It involves sequencing by synthesis ,ethod. Four enzymes DNA polymerase I, ATP sulfurylase, luciferase and apyrase are used. Apyrase degrades unused nucleotides and ATP in mixture, DNA polymerisation starts by addition of nucleotide forming base pair with sequence template. the Pi (inorganic pyrophosphate) released in reaction act as substrate for ATP sulfurylase producing ATP. ATP is used by luciferase and light is produced. this light is produced in presence of correct base pairing only.

YES, whole genome can be sequenced now a days. DNA can be fragmented in short oilgomeric forms and then sequenced further using enzymes. nowadays more efficient techniques like NEXT GEN sequencing are present which can be used for whole genome sequencing.

gladiator answered 1 year ago

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