DNA sequencing and analysis

Question 1

What is DNA sequencing?

Answer 1

• process of determining the precise order of nucleotides within a DNA molecule

Question 2

How was DNA intitially done? How is it different now?

Answer 2

• Sanger used radioactive labelled gbases and gel electrophoresis on a single gel
• fluorescent tags used which led to automation of the process
• this led to capillary sequencing version of the Sanger sequencing method that was used during the Human Genome Project

Question 3

What is the Human Genome Project?

Answer 3

• large international project where many sicentist around the world mapped the genome
  
  • making the data freely available

Question 4

How is the DNA prepared for sequencing?

Answer 4

• mixsed with a primer, DNA polymerase, an excess of normal nucleotides and terminator bases

Question 5

What happens to the mixture?

Answer 5

• pplaced in thermal cycler (PCR machine)

Question 6

WHat happens as the new strand of DNA is built ip?

Answer 6

• DNA polymerase adds nucleotides with complementary base to the signle-strand DNA template
• terminator base
  
  • when incorporated instead of a normal base, no more bases can be added
  • present in lower amounts than normal bases, added at random, results in many DNA fragments of different length, where the chain terminating bases habe neem added
• after many cycles, all of the possible DNA chain will be produced with the reaction stopped at every base

Question 7

What happens to all the DNA fragments?

Answer 7

• separated by length by capillary sequencing, which works like gel electrophoresis in minute capillary tubes
• fluorescent markers on the terminator nases used to identify the final base on each fragment
• lasers detect the different colours and thus the order of the sequence

Question 8

How is the sequence found/

Answer 8

• order of bases in the capillary tubes shows the sequence of the new, complementary strand of DNA which has been made
  
  • used to build up the sequence of the original DNA straand
• the data from the sequencing process is fed into a computer that reassembles the genomes by comparing all the fragments and finding the areas of overlap between them

Question 9

Why is the Sanger method ineffecient?

Answer 9

• difficult and time consuming

Question 10

What is the difference between the next-generation sequencing than Sanger method?

Answer 10

• uses platic slide called flow cell instead of gel or capillaries
• fragments attached and replicated in situ using PCR to form clusters of identical DNA fragments,
• still adds terminator base to stop the reaction so an image can be taken
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Question 11

why is next-gen methoid known as massively parallel sequencing?

Answer 11

as all of the clusters are sequenced and imaged at the same time, its known as massively parallel sequencing

Question 12

What are the advantages of next gen?

Answer 12

• human genome sequenced in day
• efficient and fast
• high-throughput means that the cost has fallen, so more genomes are sequenced