BIO111 - Molecular genetics - Keywords

There are 3 general types of restriction endonucleases. All of the recombinant DNA work is done with the type II enzymes. These restriction enzymes recognize short (4-8 bp), usually palindromic sequences, and cut both strands within or very close to that recognition site, leaving a 5' phosphate and a 3' OH. 

Restriction enzymes that recognize the same sequence and cut at the same position are known as isoschizomers. 

Restriction enzymes that recognize the same sequence but cut at different positions. 

A palindromic sequence is a sequence of nucleotides that, if read from 5' to 3' on one strand, matches the sequence reading from 5' to 3' on the complementary strand. 

In a blunt ended molecule, both strands terminate in a base pair. Blunt ends are not always desired in biotechnology, since when using a ligase to joint two molecules into one, the yield is significantly lower with blunt ends. 

5' and 3' overhangs are known as sticky ends, because they can base pair with complementary sequences. Sticky ends that can base pair with each other are said to have compatible ends. 

Once the base pairs have paired with each other, DNA ligase can seal the nick in the sugar-phosphate backbone, creating a covalent bond between the two. 

A clone of a gene is made by isolating and creating exact copies of just one gene of an organism. This involves copying the DNA sequence of that gene into a smaller, more easily manipulated piece of DNA, such as a plasmid. 

Cloning is the process of creating an geneticly identical replica of a donor organism or cell. 

A cloning vector is a stable, self-replicating DNA molecule to which a foreign DNA fragment can be attached for introduction into and replication in a host cell. 

An effective vector has three characteristics: 

1. An origin of replication, which allows replication in the host cell

2. One or more selectable markers, which allow the selective growth or identification of cells that have taken up the vector

3. One ore more unique sites, into which the foreign DNA can be inserted

The most common bacterial cloning vectors are plasmids. 

Plasmids are small, circular extrachromosomal DNA molecules present in many bacterial species. In the wild, plasmids often carry genes that are not essential for bacterial function, but that can play important roles in the life cycle or growth of bacteria. Their sizes range from a few kb to a few hundred kb, and they have their own origin of replication (ORI), replicating independently of the bacterial chromosome. 

They are further differentiated by the number of their copies present in the cell. Stringent plasmids are present only in one or two copies while relaxed plasmids can be present up to many dozen times. 

A cosmid is a type of hybrid plasmid. It contains a Lambda phage cos sequence and is often used as a cloning vecor in genetic engineering. 

Reverse transcriptase is an enzyme that basically does the opposite of transcription, by making a DNA copy from a RNA template. This is done because a bacteria can't process introns. In order to remove the introns, mRNA (where the introns have already been spliced out) is copied by reverse transcriptase. A cDNA (copyDNA) is created, which now consists only of exons, and which can be incorporated into a vector. 

cDNA or copy DNA is a DNA copy of an mRNA template. 

PCR is an in vitro amplication of DNA fragments.

It requires a ssDNA template, a primer, a DNA polymerase and dNTPs.

It consists of many cycles of DNA replication. In each cycle, only the region of interest is replicated, as only the oligonucleotides added by the biologist can be used as primers by the DNA polymerase. 

ddNTPs are nucleotides used for sequencing. The trick is, that they lack a -OH group on both 2' and 3' carbon atoms. This means that they can be added to a growing nucleotidechain by DNA polymerase, but they can not form a bond with the next dNTP. DNA polymerase thus stops DNA polymerization as sson as a ddNTP is added to a growing chain. ddNTPs are therefore also known as chain terminators. 

An organic molecule, consisting of a base (deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythimidine) and a 2' deoxyribose. 

If a posphatechain is added, it becomes a deoxynucleotide.

Adenine and Guanine

Cytosine and thymine

Purine, pairs with the base Thymine in DNA and Uracil in RNA

Has the following structure: 

Purine, pairs with Cytosine. 

Has the following structure: 

Pyrimidine, pairs with Guanine

Has the follwing structure: 

Pyrimidine, pairs with Adenine. Exists only in the DNA. 

Has the follwing structure: 

Nucleoside consisting of the base Adenine and a sugar: 

- 2' deoxyribose = deoxyadenosine

Nucleoside consisting of the base Guanine and a sugar

Nucleoside consisting of the base Cytosine and a sugar

Nucleoside consisting of the base Thymine and a sugar

Each phosphate in a DNA chain makes two ester bonds: 

- one with the 3' carbon of one deoxyribose

- one with the 5' carbon of the next sugar

This gives a nucleotide chain a polarity. A phosphate attached to the 5'-most sugar at one end (5') and a free -OH on the most 3' sugar at the other end of the chain (3' end). 

Connection of two bases (Adenine - Thymine (2H Bonds) and Cytosine - Guanine (3H Bonds)) via H-Bonds in the DNA helice. 

As the bases of each DNA chain are complementary to each other (Adenine - Thymine and Cytosine - Guanine), this leads to two different strands. These two strands are, as the base pairs, complementary to each other.