How the reaction products are analysed
PCR products can be visualised by
. Agarose gels areused for this. These separate DNA molecules on the basis of their
,with larger molecules moving through the pores of the gel more slowly thansmaller ones. This movement of the DNA is induced by applying anelectric current across the gel, which is submerged in buffer in a gel tank.DNA is negatively charged and moves towards a positive electrode. Afterthe DNA has been separated, it is visualised by staining with a dye thatbinds to it and fluoresces under ultraviolet light. The most commonly useddye for this purpose is called ethidium bromide.Electrophoresis is necessary to separate PCR products from the originaltemplate and also to separate marker DNA which is also loaded onto thegel. The marker DNA consists of a series of fragments of known size,against which the size of PCR product can be compared.As PCR results in the amplification of product DNA of a specific size, theamount of product on the gel is much greater than the amount of originaltemplate DNA and a strong band corresponding to the product is clearlyseen.
Reverse transcriptase polymerase chain reaction (RT-PCR)
PCR can also be used to identify mRNA targets, by a process known asthe Reverse Transcriptase Polymerase Chain Reaction. Here, in an initialstage that is not present when DNA targets are amplified, the primers bindto RNA and reversetranscriptase is used to make a cDNA copy of thetarget. Once the DNA copy has been made, the reaction can proceed asoutlined above.
A DNA fingerprint is a DNA-based pattern composed of series of bandsof different sizes. Any given individual is likely to possess a uniquecombination of bands, hence the term ‘fingerprint’. Approximately half of these bands are inherited from an individual’s mother and approximatelyhalf from their father, as illustrated in Fig 2.
Uses of DNA fingerprinting
The chances of two individuals producing identical fingerprints isvanishingly small. Consequently, DNA fingerprinting can be used to identifyindividuals. It can also be used to determine familial relationships. Althoughmuch of the usefulness of the method has been concerned with humaninvestigations, as fingerprints can be generated for other species, thetechnique has also been adopted by biologists in studying problems relatedto behaviour, population biology, taxonomy and conservation.
The theoretical basis of DNA fingerprinting
DNA fingerprinting detects the presence, throughout the genome, of DNAsequences termed
Variable Number Tandem Repeats (VNTRs).
Betweenindividuals, VNTRs vary in both their precise sequence and exact position,in that the individuals DNAs may be different. It is this variation thatcauses DNA fingerprints to be different.
The nature of VNTRs
Genomes do not just contain unique sequences of single copy DNA. Theyalso contain extensive regions in which repetitive sequences are arrangedend to end, in tandem. This is
The number of bases thatcomprise these repeated sequences varies.
containsrepetitive sequences between approximately 9 and 70 bp long, whereas in
they are generally less than 4bp long. At any givenposition
in the genome, the number of repeats in the tandemarrangement can vary. Consequently, these sequences can be referred to as
Variable Number Tandem Repeats (VNTRs).The DNA fingerprinting procedure
To produce a DNA fingerprint several steps have to be performed, asfollows:Initially, the DNA has to be extracted from a biological sample, leavingbehind any protein, carbohydrate or other materials that may also be present.The extracted DNA is then cut into a series of fragments by enzymesknown as
These recognise specific sequences in theDNA
which are generally motifs (groups) of 4 - 6 bp.When DNA is incubated with a restriction enzyme (a
the strands are cut at specific points in the vicinity of the restriction site.The pieces of DNA produced as a result of this are
A large number of fragments of different sizes are generated as a result of the restriction digest, and these are then separated by gel electrophoresis.The DNA is then transferred to a charged membrane by
To perform a Southern blot, a positively-charged membrane is placed ontop of the gel and the negatively charged DNA fragments are transferredfrom the gel to the membrane. They are then irreversibly bound to themembrane (or
as it is then called), whilst maintaining their relativepositions on the gel. Following this, the blot is subjected to
below) using a
A probe is a short piece of DNAcontaining a known sequence. For DNA fingerprinting, the sequence is therepetitive unit of the VNTR under study. The probe can synthesisedchemically and when this is done, a small number of radioactive bases canbe incorporated into the DNA, in a process known as
Through complementary base pairing, the probe will then bind onto theblot at any position where DNA of the appropriate sequence is found.This is
the position of the bound probe on the blot can be detected. In autoradiography aphotographic film is placed against the blot and the radioactivity in theprobe causes the film to be exposed.
Fig 2. DNA fingerprints of a father, mother and child showing thatthe child inherits approximately half of the DNA bands from itsfather and half from its mother. An unrelated person has a verydifferent fingerprint.
FatherMotherChildUnrelated PersonThe DNA bands aredifferent to those of theunrelated father, motherand childDNA bands are the sameas those of either themother or father
Modern Techniques in Biology: Genetics