Restriction Mapping

Recombinant DNA Technology Lecture: Restriction Mapping

In this lecture, the concept of restriction mapping is explained. Restriction mapping involves using restriction enzymes to identify the position of these restriction sites on a fragment of DNA where we don't know any information about the sequence of DNA on that fragment.

Linear vs Circular DNA Fragments

• Two scenarios are considered for genome or source of DNA in restriction mapping study: circular plasmid and linear DNA strand.

• Cutting both plasmid and linear DNA strand with one restriction enzyme results in one linear fragment.

• Cutting a circular plasmid with two restriction enzymes results in two linear fragments, while cutting a linear DNA strand with two restriction enzymes results in three fragments.

Mapping Position of Restriction Enzymes

• The process of mapping the position of restriction enzymes in a linear fragment of DNA is explained.

• Exposing a 1200 base pair fragment to HINT 3 restriction enzyme results in an 800 and 400 base pair fragment on an agarose gel.

Restriction Enzymes

This section explains how restriction enzymes work and how they can be used to map linear and circular DNA fragments.

Mapping Linear DNA Fragments

• Restriction enzymes cleave DNA at specific sites.

• When a linear fragment is restricted with two different enzymes, we can determine the position of each restriction site in relation to each other by performing a double digestion.

• A double digestion involves treating the linear fragment with both restriction enzymes at the same time and then analyzing the resulting products on an agarose gel.

• By comparing the products from single and double digestions, we can determine which restriction site is located within which fragment.

Mapping Circular DNA Fragments

• When mapping circular DNA fragments, we use restriction enzymes that cleave in just one location.

• We first restrict the plasmid with one enzyme to create a linearized fragment that can be used for further analysis.

• Double or triple digestions are performed on this linearized fragment to identify the location of each restriction site in relation to each other on the DNA strand.

Restriction Mapping

This section covers the concept of restriction mapping, its uses, and how it was done before DNA sequencing technology.

Identifying Restriction Sites

• Restriction enzymes are used to identify the position of restriction sites in a DNA fragment.

• Multiple combinations of restriction enzymes can be used to identify the position of each restriction site in relation to each other.

• This allows us to map the genome and compose an idea of the sequence of a plasmid.

Uses of Restriction Mapping

• Restriction mapping is used to identify the position of restriction sites in a DNA fragment, giving an idea of the genome sequence.

• It is also used to introduce primers for sequencing.

• Although not as commonly used today because of DNA sequencing, we still use restriction enzymes in molecular cloning.

Molecular Cloning

• Circular fragments can be cut using restriction enzymes so that additional fragments can be ligated into them.

• We will cover these concepts when discussing molecular cloning next week.