Recombinant DNA techniques

MDCAT Biology · Biology · Biotechnology. The idea in plain words, the rules to remember, the mistake to avoid and a worked example, written and checked by Mega Lecture.

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The idea

Recombinant DNA is DNA made by joining pieces from two different sources. To clone a gene, the gene is cut out with a restriction enzyme and joined into a vector, usually a plasmid, that has been cut with the same enzyme. The recombinant plasmid is put into a host cell such as E. coli. As the host multiplies, the gene is copied with it.

The polymerase chain reaction (PCR) copies a chosen stretch of DNA in a test tube, doubling it in every cycle.

Gel electrophoresis separates DNA fragments by size. DNA sequencing reads the order of the bases.

Rules to remember

Restriction enzymes (restriction endonucleases) cut DNA at specific palindromic sites, e.g. EcoRI at 5′-GAATTC-3′, often leaving sticky ends
DNA ligase seals the sugar-phosphate backbone and joins the gene to the vector
Cloning steps: isolate the gene → cut gene and plasmid with the same enzyme → join with ligase → transform host cells → select (antibiotic resistance marker) → grow
One PCR cycle: denaturation at about 94 °C → primer annealing at about 50 to 60 °C → extension by Taq polymerase at about 72 °C
Number of DNA molecules after n PCR cycles = starting number × 2ⁿ
Electrophoresis: DNA is negatively charged and moves towards the positive electrode; smaller fragments travel further
Sanger sequencing uses dideoxynucleotides, which lack a 3′-OH group and so stop the growing chain

Common mistake

Students say the largest fragment moves furthest in the gel. Large fragments are held back by the gel and stay near the wells; the smallest fragments travel furthest towards the positive electrode.

Worked example

A forensic sample contains 5 molecules of a target double-stranded DNA. How many molecules are present after 6 cycles of PCR, assuming every cycle works perfectly?

Each cycle doubles the number of molecules, so use number = starting number × 2ⁿ.
2⁶ = 2 × 2 × 2 × 2 × 2 × 2 = 64.
Number = 5 × 64 = 320.

Answer: 320 molecules of double-stranded DNA.

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