DNA Sequencing

Question 1

Shotgun Strategy Steps

Answer 1

Steps:
1. DNA extraction
2. DNA fragmentation
3. Clone into vectors
4. Transform bacteria, grow, and isolate vector DNA
5. Sequence the library
Assemble contigous fragment

Question 1

Shotgun Sequencing Phases

Answer 1

1. Start with multiple copies of a genome
2. Then there is random fragmentation typically using plasmids and bacteria and a genomic library is constructed
3. Then a strand of one of the fragments in sequences
4. The original sequence is reconstructed based on sequence overlap

Question 2

Shotgun Sequencing Overview

Answer 2

• Small and “unordered” fragments of DNA are sequenced in no particular order
• These DNA sequences are reassembled “in silico” to reconstruct the original order of sequence in genomic DNA
• Works well for small genomes (i.e. Viruses and Bacteria) that lack repetition

Question 3

Dideoxy/Sanger Sequencing Overview

Answer 3

• Uses DNA polymerase and special-chain terminating nucleotides called dideoxyribonucleoside to make partial copies of the DNA fragment to be sequenced
• The ddNTPs are derivatives of normal deoxyribonucleoside triphosphates that lack the 3’hydroxyl group and block further elongation of the strand

Question 4

Manual Dideoxy Sequencing Steps

Answer 4

-Add labeled DNA primer
-Add excess amounts of normal dNTPs
-Add DNA polymerase and divide into 4 separate tubes
-Add a small amount of chain-terminating ddNTP to each tube

Question 5

Manual Dideoxy Sequencing Phases

Answer 5

• A single-stranded DNA fragment is first hybridized with a short, fluorescently labeled, DNA primer
• DNA polymerase and an excess of A, C, G, or T are added to primed DNA and divided into 4 tubes
• Chain terminating A, C, G, or T is added to each tube
• Each reaction produces a set of DNA copies that terminate at a different point in the sequence
• These are then separated into 4 different lanes of gel electrophoresis labeled A, C, G, or T
• The formed bands represent fragments that have terminated at a given nucleotide but at a different position in the DNA
• Once the bands are read in order going from the bottom to the top, the sequence of the newly synthesized strand can be determined
• The new sequence is complementary to the orignal strand

Question 6

Automated Dideoxy Steps Steps

Answer 6

• start with a mix of DNA products each containing a chain-terminating ddNTP labeled with a different fluorescent marker
• The product is loaded into a capillary gel and undergo electrophoreis
• Size separated products are read in sequence

Question 7

Automated Dideoxy Sequencing Phases

Answer 7

• An excess amount of normal dNTPs plus a mixture of 4 different chain terminating ddNTPs which is each labeled with a differently colored fluorescent tag
• The products are loaded into a long, thin capillary gel and are separated by electrophoresis
• A camera reads the color of each band as it moves through the gel and a computer assembles the sequence

Question 8

Illumina Sequencing

Question 9

Second Gen Sequencing Overview

Answer 9

• Instead of using bacteria to generate cell libraries, these use PCR amplification of billions of DNA fragments
• The PCR-generated copies remain bound in proximity to the original DNA fragment instead of floating away in the solution
• This process generates clusters, each containing about 1000 identical copies of a small bit of the genome
• The clusters are then sequenced at the same time

Question 10

Illumina Sequencing Overview

Answer 10

• Based on the dideoxy method
• A nucleotide is attached to a removable fluorescent molecules
• A chain-terminating chemical adduct is also added, instead of a 3’-OH group as in conventionally dideoxy sequencing.
• With the addition of the chain-terminating adduct, the nucleotides carry a chemical group that blocks elongation by DNA polymerase and can be removed chemically
• Billions of sequencing reactions are carried out simultaneously
• The DNA sequence is determined by the sequence of colour changes it undergoes as the elongation reaction proceeds

Question 11

Illumina Sequencing Phases

Answer 11

• A nucleotide is attached to a removable fluorescent molecule and chain-terminating adduct
  The four fluorescently labeled nucleotides and the DNA polymerase are added to billions of immobilized DNA clusters
• Only the appropriate complementary nucleotide is covalently incorporated at each cluster
• The unicorporated nucleotideas are washed away
• A camera takes an image of which of the four nucleotides was added to the chain at each cluster
• The fluorescent label and the 3’-OH blocking group are removed enzymatically and washed away
• The process is repeated many times
• By keeping tracj of the colour cahnges at ecah cluster, the sequence can be read