M6: DNA SEQUENCING

Question 1

this is the process of determining the exact order of nucleotides within a DNA molecule

Answer 1

DNA sequencing

Question 2

DNA sequencing has been feasible because of what developments ?

Answer 2

Restriction endonucleases

Gel electrophoresis

PCR techniques

DNA techniques

Question 3

What are the two basic techniques for DNA sequencing ?

Answer 3

Chemical sequencing (Maxam and Gilbert)

Enzymatic sequencing (Sanger)

Question 4

What is the principle of the Maxam and Gilbert procedure ?

Answer 4

Based on the chemical modification of DNA and subsequent cleavage at specific bases

Question 5

Maxam-Gilbert sequencing requires ____ 5’ end or 3’ of the DNA followed by ____ of the DNA fragment to be sequenced

Answer 5

radioactive labelling

purification

Question 6

What is the steps for the Maxam-Gilbert sequencing ?

Answer 6

Radioactive labelling of one end of the DNA fragment to be sequenced by a kinase reaction using 32P

Cut the DNA fragment with the specific restriction enzyme, resulting in two unequal DNA fragments

Denature the double-stranded DNA to single-stranded DNA by increased temperature

Cleave the DNA strand at specific positions using chemical reactions (Modification Step)

Chemical treatment generates breaks at the four nucleotide bases in the four reaction mixtures (G, A+G, C, and C+T)

Question 7

___ selectively attacks purine (A and G)

Answer 7

Dimethyl sulphate

Question 8

___ selectively attacks pyrimidines (C and T)

Answer 8

Hydrazine

Question 9

This reagent breaks the DNA chain after guanine base

Answer 9

Reagent G

Question 10

This reagent breaks the DNA chain after adenine and guanine bases

Answer 10

Reagent A+G

Question 11

This reagent breaks the DNA chain after cytosine base

Answer 11

Reagent C

Question 12

This reagent breaks the DNA chain after cytosine and guanine bases

Answer 12

Reagent C+T

Question 13

The concentration of the modifying chemicals is controlled to introduce, on an average, ___. Thus a series of ___ is generated, starting from the radiolabeled end of the first “cut” site in each molecule.

Answer 13

one modification per molecule

labelled fragments

Question 14

Fragments are subjected to ___ in high-resolution ___ for size-based separation

Answer 14

electrophoresis

acrylamide gels

Question 15

To visualize the fragments, the gel is exposed to ___ for ___ , which yields a series of dark bands, each corresponding to a ___, from which the nucleotide sequence may be inferred

Answer 15

X-ray film

autoradiography

radiolabeled DNA fragment

Question 16

Where is the location of large and small fragments in reading the gel of maxam-gilbert sequence and why ?

Answer 16

largest at the bottom and smallest at the top

larger the fragment, the more it is slowed down by the gel

Question 17

This sequencing method dominated the DNA sequencing platform, bringing successful completion of the Human Genome Project

Answer 17

Sanger sequencing

Question 18

Although this technique has been replaced by ___, it is still used today for ___

Answer 18

next generation sequencing methods

smaller-scale projects

Question 19

In order to perform the sequencing, one must first convert ___ DNA into ___ DNA. This can be done by ___ the double stranded DNA with ____

Answer 19

dsDNA
ssDNA

NaOH

Question 20

In the sanger sequencing method at least one of the four dNTPs should be ___ in each of the four reaction mixtures tubes in order to permit the ___ of DNA bands after the gel electropheresis

Answer 20

radioactive

autoradiographic development