DNA Cloning and Restriction Enzymes in Genomics

DNA cloning

The mass production of identical copies of a segment of DNA.

Cloning vector

An artificially constructed DNA molecule that can replicate in a host organism, e.g. a modified bacterial plasmid.

Recombinant DNA

Foreign DNA combined with a vector.

Transformation

The introduction of recombinant DNA into a host.

Restriction enzymes (RE)

Enzymes that recognize and cut a specific DNA sequence.

Palindromic sequence

A sequence that reads the same on the top strand (5'®3') as it does on the bottom (5'®3').

Staggered cuts

Cuts made by some REs that give overhanging or sticky ends.

Flush cuts

Cuts made by some REs that give blunt ends.

Methylation

The process by which restriction sites in bacterial DNA are protected from REs.

EcoRI

A specific restriction enzyme named after Escherichia coli strain RY13.

Frequency of occurrence of RE sites

The number of cuts a RE makes in a DNA molecule depends on how often the recognition sequence occurs.

Probability of a specific base

In an organism with random distribution of bases, the probability of a specific base at a given position is 1⁄4.

Frequency of a particular restriction site

Calculated as (1⁄4)n where n is the number of base pairs in the recognition sequence.

Example of Sau3AI

Recognizes the sequence GATC.

Ligation

The joining of two DNA fragments, forming phosphodiester bonds.

Blunt-ended DNA

DNA fragments that can be ligated together, although it is harder for them to find each other.

Ligase

An enzyme that joins DNA fragments and forms phosphodiester bonds.

Applications of cloned DNA

Includes mapping genes, sequencing, mutating, transforming cells, making proteins, and studying its function.

Single-stranded overhangs

Can be at either the 5' or the 3' end of DNA fragments.

Cloning procedure step 1

Isolate DNA from an organism.

Cloning procedure step 2

Use restriction enzymes to cut DNA into fragments.

Cloning procedure step 3

Ligate fragments into a cloning vector.

Cloning procedure step 4

Introduce recombinant DNA into a host.

Cloning procedure step 5

The host will replicate the DNA and pass copies to its progeny.