Question

In vitro DNA synthesis using the Klenow fragment (the large fragment)

Experimental Uses:
a. Fill-in reaction to label ___ ends of DNA. It can also be used to create a blunt-end for cloning.

b. DNA ____ (must use Klenow)

Answer 1

The Klenow fragment is used in vitro DNA synthesis for labeling DNA ends and in DNA sequencing as part of the Sanger method. It allows for the creation of blunt ends for cloning and is integral in amplifying DNA sequences through PCR, akin to DNA xeroxing.

Step-by-step explanation:

In vitro DNA synthesis using the Klenow fragment, a modified version of DNA polymerase I, allows for a variety of applications:

• The fill-in reaction is used to label the 5' or 3' ends of DNA. This is important for several molecular biology techniques, including adding labels to DNA for visualization or for creating blunt ends ready for cloning.
• The Klenow fragment also plays a crucial role in the Sanger method for DNA sequencing. In this process, it elongates DNA strands by adding nucleotides one by one. When a dideoxynucleotide is incorporated, which lacks a 3'-OH group, the chain elongation is terminated, resulting in DNA fragments of various lengths. These fragments can then be analyzed to determine the DNA sequence.

Further uses include gene synthesis for cloning and acting as a template for Polymerase Chain Reaction (PCR), often referred to as 'DNA xeroxing' due to the way it amplifies DNA sequences.