Biotechnology: Processes

DNA Tools, Techniques and Vocabulary

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Christian Bien Portrait_edited.jpg

Ben Whitten

Learning Objectives

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What are the DNA tools used in biotechnology?
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1. Restriction enzymes: These are enzymes that cut DNA molecules at recognition sites (specific nucleotide sequences), usually 4 to 8 nucleotide bases long. Naturally occurring restriction enzymes protect bacteria by cutting foreign DNA and removing invading organisms. There are two types of cuts from restriction enzymes: sticky ends, where strands have overhanging complementary bases and are specific, and blunt ends, where there are no overhanging nucleotides and they are unspecific.


2. DNA ligase: This enzyme uses complementary base-pairing rules to seal and reassemble DNA strands in the process of ligation. DNA ligase catalyses a strong covalent bond, closing up the sugar-phosphate backbone to hold the two strands of DNA together. This is useful for recombinant DNA technology.


3. DNA polymerase: This is a class of enzymes that synthesise new strands of DNA based on a template strand and according to complementary base-pairing rules. DNA polymerase adds free nucleotides one at a time. As it moves along a template strand, it attaches the complementary nucleotide to make a new strand. This is used in amplifying DNA during PCR.


4. Primers: Short fragments of single-stranded DNA or RNA. Primers assist in synthesising new strands of DNA by acting as a signal for the polymerase to begin synthesis.

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What are the main DNA techniques and vocabulary in biotechnology?
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1. Annealing: This is the process of when two pieces of DNA as joined/hybridised via complementary base pairing rules (from overhanging sticky ends). The two pieces are joined by weak hydrogen bonds only, and therefore temporarily. It is a biochemical process of bonding two segments of DNA at an optimal temperature of 50-60ºC.


2. Amplifying DNA: To significantly increase the number of copies of a DNA sequence. This can be achieved either in vivo by inserting the sequence to be amplified into a cloning vector that can be replicated within a host cell or in vitro by PCR.


3. DNA ladder: A collection of DNA fragments known as base pair lengths. A ladder is a standard/a set of molecular size markers used to compare the sample DNA with known DNA and is used in electrophoresis.


4. STRs (Short Tandem Repeats): Sequences of DNA that repeat a certain number of times. They are highly variable segments of DNA, typical of non-coding and non-regulatory DNA that occur throughout the genome and contain repeats of the same sequence of several nucleotides, for example, CTACTACTACTA etc. The number of times the sequence is repeated is unique in different individuals.


5. Agarose gel: A three-dimensional matrix formed of helical agarose molecules in supercoiled bundles aggregated into three-dimensional structures with channels and pores through which biomolecules can pass (nucleic acids, proteins etc.)


6. Micropipette: A tool that dispenses small amounts of samples into PCR tubes or the wells of an electrophoresis gel. The volume is adjustable to the microlitre, and the tips are removed after every use to avoid contamination.

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