Instruction 8 - DNA Vectors and Restriction Enzymes

Extrachromosomal DNA molecules found in bacteria and some eukaryotes, typically circular and smaller than bacterial chromosomes. They replicate independently and can carry genes for antibiotic resistance or other traits.

A specific DNA sequence recognized and cut by a restriction enzyme, often palindromic, meaning it reads the same backward and forward.

Enzymes that recognize and cleave specific DNA sequences, acting like molecular scissors to cut DNA at precise locations.

The process of using restriction enzymes to cut DNA at specific sites, creating fragments that can then be analyzed or manipulated in various ways. This is a fundamental technique in molecular biology and genetic engineering.

A sequence of DNA that initiates replication of a DNA molecule, often found on plasmids and bacterial chromosomes.

The transfer of genetic material between bacteria, often mediated by plasmids, allowing for rapid spread of genetic information.

Mutations in the restriction-modification system of bacteria, preventing them from cutting their own DNA but retaining the ability to methylate it, making them useful for cloning experiments.

The process of inserting a gene into a plasmid, creating a recombinant molecule that can be amplified and expressed in a host cell.

A type of restriction enzyme that recognizes a specific palindromic sequence and cuts the DNA at a specific location within or near the recognition site. These enzymes are widely used in molecular cloning due to their predictable and precise cutting action.

A group of restriction enzymes that cleave DNA at random locations far away from their recognition sequence. They often contain a combination of restriction and modification enzymes.

A subtype of restriction enzymes that cut DNA at a specific distance from the recognition sequence. They typically cut on one side of the recognition sequence, always at a specific distance.

A type of restriction enzyme that requires two non-palindromic sequences with opposite orientations to be near each other on the same DNA molecule for recognition. They cut DNA at random locations, but only after recognizing the two specific sequences.

The naming convention for restriction enzymes. It typically includes the genus of the bacteria, the species, the strain (if applicable), and a Roman numeral representing the order of discovery in that strain.

A sequence that reads the same backward as forward, but on opposite strands of DNA. Restriction enzymes often recognize palindromic sequences to cut DNA.

The average length of DNA fragments generated by a restriction enzyme depends on the length of the palindromic sequence it recognizes. Longer sequences are less frequent in the genome, leading to larger fragments.

The process of using restriction enzymes to cut DNA at specific locations. This technique is crucial for genetic engineering and molecular cloning.

The process of separating DNA fragments based on their size using an electric current through a gel matrix. Smaller fragments travel faster through the gel.

Plasmids are small, circular DNA molecules found in bacteria, often used as vectors in cloning. They can carry genes for antibiotic resistance, allowing for the selection of bacteria that have taken up the plasmid.

The process of incorporating foreign DNA into a host cell, typically bacteria, to create a recombinant organism.

The ability of a restriction enzyme to cut DNA into fragments with either blunt or sticky ends.

A DNA fragment with single-stranded overhangs, resulting from a staggered cut by a restriction enzyme.

A DNA fragment without any single-stranded overhangs, resulting from a straight cut across the DNA by a restriction enzyme.

An enzyme that joins DNA fragments together by forming a phosphodiester bond between the 5'-phosphate group of one fragment and the 3'-hydroxyl group of another fragment.

A fragment of DNA polymerase that lacks exonuclease activity but retains polymerase activity, used to fill in single-stranded overhangs.

A restriction enzyme used in molecular biology to cut DNA at a specific sequence, generating DNA fragments that can be analyzed.

A restriction enzyme used in molecular biology to cut DNA at a specific sequence, generating DNA fragments that can be analyzed.

A restriction enzyme used in molecular biology to cut DNA at a specific sequence, generating DNA fragments that can be analyzed.

A technique used to separate DNA fragments based on their size, revealing their lengths and allowing for analysis of the DNA sequence or identification of specific fragments.

The orientation of the OVA-GFP insert is the same as the CMV promoter. This means the insert is in the same direction as the CMV promoter.

The orientation of the OVA-GFP insert is opposite to the CMV promoter. The insert is in the opposite direction to the CMV promoter.

When selecting restriction enzymes for analysis, we must consider the following:

1. The specificity of the chosen enzyme - ensuring it cuts at the desired restriction site.
2. The compatibility of the chosen enzyme with the target DNA - ensuring the sequence recognized by the enzyme is present in the target DNA.
3. The number of restriction sites within the target DNA - selecting an enzyme with an appropriate number of sites for the desired outcome.
4. The location of the restriction sites - ensuring they are in the desired location for the chosen cloning strategy.
5. The availability of the chosen enzyme - making sure the enzyme is easily accessible and affordable.

The mass of a DNA fragment is determined by the difference in distance, measured in base pairs (bp), between the sites where the restriction enzyme cuts. The distance between the sites is determined by subtracting the smaller number from the larger one. Consider the following for calculations:

1. When the cutting sites are at the beginning and end of a sequence: Subtract the smaller distance from the larger one. Example: Cutting sites are at 1 bp and 100 bp, resulting in a fragment mass of 100-1 = 99 bp.
2. When calculating the masses of fragments within a larger sequence, measure the distance between two consecutive cutting sites and subtract the smaller number from the larger one. Example: Cutting sites are at 10 bp, 20 bp, and 50 bp. The fragment mass between the first two cutting sites is 20-10 = 10 bp. The fragment mass between the second and third cutting sites is 50-20 = 30 bp.

Closed inserts are circular fragments of DNA that have been inserted into a plasmid. These inserts are incompatible with the host cell because circular DNA is not always recognized by the replication machinery or the repair system. The circular insert can be digested, by the cell, and recycled. Also, the host cell may destroy the closed inserts containing toxic enzymes. This results in the closed insert being lost or unable to be duplicated within the host.