Unit II: r-DNA technology I

Outlines of cloning strategies

Gene cloning
Gene cloning involves separation of specific gene or DNA fragments from a donor cell, attaching it to small
carrier molecule called vector and then replicating this recombinant vector into a host cell.
Steps involved in gene cloning
• Isolation of donor DNA fragment or gene
• Selection of suitable vector
• Incorporation of donor DNA fragment into the vector
• Transformation of recombinant vector into a suitable host cell
• Isolation of recombinant host cell

1. Isolation of donor DNA fragment or gene

At first a donor DNA fragment should be isolated. There are two method for isolation of desired gene or DNA
fragment.
Using restriction endonuclease enzyme: the enzyme restriction endonuclease is a key enzyme in molecular
gene cloning. It has specific restriction site for its action. The enzyme RE generates a DNA fragment either
with blunt end or with sticky end
Using reverse transcriptase enzyme: reverse transcriptase enzyme Synthesizes complementary DNA strand of
the desired gene using its mRNA.
2. Selection of suitable cloning vector:
When donor DNA fragment is incorporated into a host cell, it will not replicates because the isolated gene do
not have the capacity to replicated itself. So before introduction of donor fragment into host, a suitable vector
should be selected.
Cloning vector is the DNA molecule capable of self-replication inside the host cell. the main function of
cloning vector is to replicates the inserted DNA fragment inside the host cell.
Examples of cloning vectors: Plasmid, BAC, YAC, Λ-bacteriophage, expression vectors etc.
Characteristics of a cloning vectors
It must be self-replicating inside host cell
It must possess restriction site for RE enzymes
Introduction of donor DNA fragment must not interfere with replication property of the vector
It must possess some marker gene such that it can be used for later identification of recombinant cell.
3. Incorporation of donor DNA fragment with Plasmid vector:
The plasmid vector is cut open by the same RE enzyme used for isolation of donor DNA fragment
The mixture of donor DNA fragment and plasmid vector are mixed together.
In the presence of DNA ligase, base pairing of donor DNA fragment and plasmid vector occurs forming
recombinant vector in the mixture.
4. Transformation of recombinant vector into suitable host:
The recombinant vector is transformed into suitable host cell. ie bacterial cell
Some bacteria are naturally transformable, they take up the recombinant vector automatically. For
examples: Bacillus, Haemophillus, Helicobacter pylori, are naturally competent
Some other bacteria are not naturally competent, in those bacteria recombinant vector are incorporated by
artificial method such as Ca++ ion treatment, electroporation etc.
5. Isolation of recombinant cell:
The recombinant host cell is then grown in culture media but the culture may contains colonies both
recombinant cell and non-recombinant cell.
For isolation of recombinant cell from non-recombinant
cell, marker gene of plasmid vector is employed.
For examples, PBR322 plasmid vector contains different marker gene (Ampicillin resistant gene and
Tetracycline resistant gene. When pst1 RE is used it knock out Ampicillin resistant gene from the plasmid, so
that the recombinant cell become sensitive to Ampicillin.

DNA sequencing- Maxam Gilbert and Sanger’s methods

Maxam–Gilbert sequencing is a method of DNA sequencing developed by Allan Maxam and Walter
Gilbert in 1976–1977.
 Maxam–Gilbert sequencing requires radioactive labeling at one 5′ end of the DNA fragment to be
sequenced (typically by a kinase reaction using gamma-32P ATP) and purification of the DNA.
 Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in
each of four reactions (G, A+G, C, C+T).
 For example, the purines (A+G) are depurinated using formic acid, the guanines (and to some extent
the adenines) are methylated by dimethyl sulfate, and the pyrimidines (C+T) are hydrolysed using
hydrazine.
 The addition of salt (sodium chloride) to the hydrazine reaction inhibits the reaction of thymine for the
C-only reaction.
 The modified DNAs may then be cleaved by hot piperidine;(CH2)5NH at the position of the modified
base.
 The concentration of the modifying chemicals is controlled to introduce on average one modification
per DNA molecule.
 Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each
molecule.
  The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for
size separation.
 To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of
dark bands each showing the location of identical radiolabeled DNA molecules. From presence and
absence of certain fragments the sequence may be inferred.

Sangers method of DNA sequencing

 In the enzymatic Sanger dideoxy procedure, the sequence is determined by making a copy of the
single-stranded DNA, using DNA polymerase.
 This enzyme uses deoxyribonucleoside triphosphates (dNTPs) as substrates and adds them to a primer.
The primer is hydrogen bonded to the 3' end of the DNA to be sequenced.
 The DNA with the primer is divided into four separate reaction mixtures. Each reaction mixture
contains all four dNTPs and in addition, one of the four dideoxy analogs (dideoxyribonucleoside
triphosphates ddNTPs) of the deoxyribonucleoside triphosphates.
 Because in the dideoxy sugar the 3'-hydroxyl has been replaced by a hydrogen, continued extension of
the chain cannot occur.
 The dideoxy analog thus acts as specific chain-termination reagent. Fragments of variable length are
obtained depending on the ddNTP in the mixture.
 The formed nucleic acid fragments are visualizes by using either a labelled (radioactive or fluorescent)
primer or dNTPs.
 Tools of r-DNA technology: Enzymes- Restriction endonucleases and ligases

Restriction endonucleases
A restriction enzyme or restriction endonuclease is an enzyme that cleaves DNA into fragments at or near
specific recognition sites within the molecule known as restriction sites. Restriction enzymes are commonly
classified into four types, which differ in their structure and whether they cut their DNA substrate at their
recognition site, or if the recognition and cleavage sites are separate from one another. A restriction enzyme
will make a double-stranded cut in the DNA molecule. These enzymes are found in bacteria and archaea and
provide a defense mechanism against invading viruses.
Type l
 Type I restriction enzymes were the first to be identified and were first identified in two different
strains (K-12 and B) of E. coli.
 These enzymes cut at a site that differs, and is a random distance (at least 1000 bp) away, from their
recognition site. The cofactors S-Adenosyl methionine (AdoMet), hydrolyzed adenosine triphosphate
(ATP), and magnesium (Mg2+) ions, are required for their full activity.
 Type I restriction enzymes possess three subunits called HsdR, HsdM, and HsdS;
 HsdR is required for restriction; HsdM is necessary for adding methyl groups to host DNA
(methyltransferase activity), and HsdS is important for specificity of the recognition (DNA-binding)
site in addition to both restriction (DNA cleavage) and modification (DNA methyltransferase) activity.

Type II
Typical type II restriction enzymes recognize and cleave DNA at the same site, and they do not use ATP or
AdoMet for their activity—they usually require only Mg2+ as a cofactor.
It can either cleave at the center of both strands to yield a blunt end. Or it can cleave at a staggered position
leaving overhangs called sticky ends.
 Type IIB restriction enzymes (e.g., BcgI and BplI) are multimers, containing more than one
subunit. They cleave DNA on both sides of their recognition to cut out the recognition site. They
require both AdoMet and Mg2+ cofactors.
 Type IIE restriction endonucleases (e.g., NaeI) cleave DNA following interaction with two copies of
their recognition sequence. One recognition site acts as the target for cleavage, while the other acts as
an allosteric effector that speeds up or improves the efficiency of enzyme cleavage.
 Similar to type IIE enzymes, type IIF restriction endonucleases (e.g. NgoMIV) interact with two copies
of their recognition sequence but cleave both sequences at the same time.
 Type IIG restriction endonucleases (e.g., Eco57I) do have a single subunit, like classical Type II
restriction enzymes, but require the cofactor AdoMet to be active.
 Type IIM restriction endonucleases, such as DpnI, are able to recognize and cut methylated DNA.
 Type IIS restriction endonucleases (e.g., FokI) cleave DNA at a defined distance from their non-
palindromic asymmetric recognition sites.
 Similarly, Type IIT restriction enzymes (e.g., Bpu10I and BslI) are composed of two different subunits.
Some recognize palindromic sequences while others have asymmetric recognition sites

Type III

 Type III restriction enzymes (e.g., EcoP15) recognize two separate non-palindromic sequences that are
inversely oriented.
 They cut DNA about 20–30 base pairs after the recognition site. These enzymes contain more than one
subunit and require AdoMet and ATP cofactors for their roles in DNA methylation and restriction,
respectively.
 Type III enzymes recognize short 5–6 bp-long asymmetric DNA sequences and cleave 25–27
bp downstream to leave short, single-stranded 5' protrusions.
Type IV
Type IV enzymes recognize modified, typically methylated DNA and are exemplified by the McrBC and Mrr
systems of E. coli
Type V
Type V restriction enzymes (e.g., the cas9-gRNA complex from CRISPRs utilize guide RNAs to target
specific non-palindromic sequences found on invading organisms. They can cut DNA of variable length,
provided that a suitable guide RNA is provided.

Ligases
A ligase is an enzyme that can catalyze the joining of two large molecules by forming a new chemical bond,
usually with accompanying hydrolysis of a small pendant chemical group on one of the larger molecules or the
enzyme catalyzing the linking together of two compounds. In general, a ligase catalyzes the following reaction:
Ab + C → A–C + b
or sometimes
Ab + cD → A–D + b + c + d + e + f
where the lowercase letters signify the small, dependent groups. Ligase can join two complementary
fragments of nucleic acid and repair single stranded breaks that arise in double stranded DNA during
replication.

Restriction mapping

 Restriction mapping is a method used to map an unknown segment of DNA by breaking it into pieces
and then identifying the locations of the breakpoints.
 This method relies upon the use of proteins called restriction enzymes, which can cut, or digest, DNA
molecules at short, specific sequences called restriction sites.
 After a DNA segment has been digested using a restriction enzyme, the resulting fragments can be
examined using a laboratory method called gel electrophoresis, which is used to separate pieces of
DNA according to their size.
 One common method for constructing a restriction map involves digesting the unknown DNA sample
in three ways.
 Here, two portions of the DNA sample are individually digested with different restriction enzymes, and
a third portion of the DNA sample is double-digested with both restriction enzymes at the same time.
 Next, each digestion sample is separated using gel electrophoresis, and the sizes of the DNA fragments
are recorded.
 The total length of the fragments in each digestion will be equal. However, because the length of each
individual DNA fragment depends upon the positions of its restriction sites, each restriction site can be
 mapped according to the lengths of the fragments.
 The information from the double-digestion is particularly useful for correctly mapping the sites. The
final drawing of the DNA segment that shows the positions of the restriction sites is called a restriction
map.

Cloning vectors- Plasmids, Cosmids, and λ phages

A vector is a molecule of DNA which can replicate inside the host cell and is maintained in a stable and
characteristic number of copies inside the cell. A large number of cloning vectors such as
plasmids, bacteriophages, cosmids, BACs and YACs are available. Many factors such as the size of insert,
copy number of vectors (which affects the yield of the recombinant DNA)and the method to be used for
cloning should be considered while selecting the cloning vector.
1. Plasmids
pBR322
 The pBR322 plasmid contains a gene that allow the bacteria to be resistant to the antibiotics
tetracycline and amipicillin. The p stands for "plasmid," and BR for "Bolivar" and "Rodriguez."
 To use pBR322 plasmid to clone a gene, a restriction endonuclease first cleaves the plasmid at a
restriction site.
 pBR322 plasmid contains many restriction sites few of which are PstI, SalI HindIII, BamHI and EcoRI.
The PstI restriction site is located within the gene that codes for ampicillin and SalI HindIII, BamHI in
tetracycline resistance.
 Cleaving at either restriction site will inactivate their respective genes and antibiotic resistance. The
target DNA is cleaved with a restriction endonuclease at the same restriction site.
 The target DNA is then annealed to the plasmid using DNA ligase. After the target DNA is
incorporated into the plasmid, the host cell is grown in a environment containing ampicillin or
tetracycline, depending on which gene was left active. Many copies of the target DNA is created once
 the host is able to replicate.

pUC18
 pUC18- UC stands for University of California. The main feature of this vector is presence of multiple
cloning sites. pUC18 plasmid contains a gene that makes the host cell ampicillin resistant.
 It also contains a gene that allows it produce beta-galactosidase, which is an enzyme degrades certain
sugars. The enzyme produces a blue pigment when exposed to a specific substrate analog.
 This allows the host to be readily identified. The gene for beta-galactosidase contains a polylinker
region that contains several restriction sites.
 The pUC18 plasmid can be cleaved by several different restriction endonucleases which provide more
versatility.
 When the polylinker sequence is cleaved and the target DNA is introduced and ligased, this inactivates
the gene that codes for beta-galactosidase and the enzyme will not be produced. The host cell will not
produce a blue pigment when exposed to the substrate analog. This allows the recombinant cells to be
readily identified and isolated.

Ti Plasmid
 A Ti or tumour inducing plasmid is a plasmid that often, but not always, is a part of the genetic
equipment thatAgrobacterium tumefaciens and Agrobacterium rhizogenes use to transduce their genetic
material to plants.
 The Ti plasmid is lost when Agrobacterium is grown above 28 °C. Such cured bacteria do not induce
crown galls, i.e. they become avirulent. pTi and pRi share little sequence homology but are functionally
rather similar.
 The Ti plasmids are classified into different types based on the type of opine produced by their genes.
The different opines specified by pTi are octopine, nopaline, succinamopine and leucinopine.
 The T-DNA carries enzymes that convert plant metabolites to hormones cytokinin and auxin, which
stimulate tumor formation. It also carries genes for opine synthesis. Opines serve as a food for the
bacterium and are synthesized in higher concentrations in tumors cells
 Researchers engineered this Ti plasmid by disrupting these tumor causing and opine synthesis genes
and replaced them with selectable marker genes conferring resistance to antibiotics. Ti plasmid based
vectors were constructed with the T-DNA carrying following components ori for origin of replication,
allowing the plasmid to replicate; (ii)Right border sequence which is necessary for transfer of Ti
plasmid into plant genome; (iii)A multiple cloning site to ease the insertion of gene of interest into the
region between T-DNA border sequences.
 Recombinant genes can then be integrated into the T-DNA of the Ti plasmid, and the plasmid can be
used to infect plant cells. The infected cells are placed on a culture medium containing growth factors
and the selectable antibiotic. Only the cells harboring T-DNA can grow in the presence of the
antibiotic.

2. Cosmids

 Cosmids are plasmids that incorporate a segment of bacteriophage λ DNA that has the cohesive end site
(cos) which contains elements required for packaging DNA into λ particles.
 It is normally used to clone large DNA fragments between 28 and 45 Kb. The efficiency of cloning
decreases in plasmids as fragment size increases and the length of non-essential region in λ phage
vectors limits the length of inserted DNA in them.
 The term cosmid includes ‘cos’ from cos sites and ‘mid’ from plasmid vectors as these are plasmid
vectors containing cos sites of λ phage vectors.
 Cosmids are circular double-stranded DNA molecules containing a prokaryotic origin of replication, a
selection marker, cloning sites for restriction endonucleases and λ cos sites.
 They are small (4-6 Kb) and can hold insert DNA fragments up to 47 Kb in length. Cosmid vectors are
used for cloning in a manner essentially similar to plasmid vectors and can clone larger DNA fragments
than plasmid or phage.
 Insert DNA is ligated between two cos sites using restriction endonucleases. DNA is packaged in
vitro and introduced into E. coli. Since they lack phage genes, they act as plasmids when transferred
into E. coli. Inside the bacterial cell, the cosmid circularizes and replicates as a plasmid maintaining 15-
20 copies per cell.
 Yeast genome was sequenced using cosmid vectors. The cosmids require tedious working protocols for
cloning and screening. Another disadvantage of cosmids is that their enormous insert size can result in
recombination within the cloned sequences. But once desired recombinant clone is identified, isolation
of DNA is easy.
 3. lamda phages

Lambda bacteriophages are the most important and widely used vectors having linear genomes. The linear
genome bears short (12 bp) single-stranded sequences at its ends known as cos sites which are complementary
to each other. These cohesive or ‘sticky’ ends enable circularization of the λ genome after infection during
packaging.

Phage vectors bind to receptors on the bacterial surface. This is known as adsorption. The phage DNA is
injected into the cell and the phage life cycle can start.

The phage genome circularizes and the phage can initiate either the lytic or lysogenic cycle, depending on
nutritional and metabolic status of the host cell. The ratio of phage to bacteria during adsorption also influences
the lytic or lysogenic growth cycle. Specific environmental changes can also trigger the lysis of host cell.

Important features contributing to the utility of λ phages as vectors are:

1. Only two-thirdsof the genome is essential and about one-third of the genome is non-essential which can
be discarded and replaced with foreign DNA. The middle region of the lambda genome is dispensable
as it controls the lysogenic properties of the phage, therefore this region can be deleted without
disrupting the functions required for the lytic growth cycle. This region is substituted with the
polylinker.
2. The packaging mechanism in λ phage facilitates insertion of recombinant DNA as packaging into
infectious phage particles is possible only if DNA is between 40 to 53 Kb long. λ bacteriophage vectors
can be digested to release three fragments, of which two fragments harbor essential genes comprising ~
30 Kb. The third middle fragment can be discarded when additional DNA is inserted for cloning,
producing viable phage particles. Therefore bacteriophage vectors allow the insertion of DNA
fragments of up to 23 Kb.

There are two kinds of λ phage vectors - insertion vector and replacement vector. Insertion vectors contain a
unique cleavage site whereby foreign DNA with size of 5–11 kb may be inserted. In replacement vectors, the
cleavage sites flank a region containing genes not essential for the lytic cycle, and this region may be deleted
and replaced by the DNA insert in the cloning process, and a larger sized DNA of 8–24 kb may be inserted.

Hosts- E.coli

 Bacteria make useful tools for genetic research because of their relatively small genome size compared
to eukaryotes.
 E. coli cells only have about 4,400 genes and live their entire lifetime in a haploid state, with no second
allele to mask the effects of mutations during protein engineering experiments.
 Bacteria typically grow much faster than more complex organisms. E. coli grows rapidly at a rate of
one generation per twenty minutes under typical growth conditions.
  This allows for getting the genetic experimental results in mere hours instead of several days, months
or years.
 E. coli is the most highly studied microorganism and an advanced knowledge of its protein expression
mechanisms makes it simpler to use for experiments where expression of foreign proteins and selection
of recombinants is essential.
 E. coli is readily transformed with plasmids and other vectors, easily undergoes transduction, and
preparation of competent cells (cells that will take up foreign DNA) is not complicated .

Molecular markers–RFLP, AFLP and RAPD

 A molecular marker is a molecule contained within a sample taken from an organism (biological
markers) or other matter.
 It can be used to reveal certain characteristics about the respective source. DNA, for example, is a
molecular marker containing information about genetic disorders and the evolutionary history of life.
 Specific regions of the DNA (genetic markers) are used for diagnosing the autosomal recessive genetic
disorder cystic fibrosis, taxonomic affinity (phylogenetics) and identity (DNA barcoding)
 In genetics, a molecular marker (identified as genetic marker) is a fragment of DNA that is associated
with a certain location within the genome. Molecular markers are used in molecular biology and
biotechnology to identify a particular sequence of DNA in a pool of unknown DNA.

1. Restriction fragment length polymorphism (RFLP)

Restriction fragment length polymorphism (RFLP) is a technique that exploits variations
in homologous DNA sequences, known as polymorphisms, in order to distinguish individuals, populations, or
species or to pinpoint the locations of genes within a sequence.
The term may refer to a polymorphism itself, as detected through the differing locations of restriction enzyme
sites.
In RFLP analysis, a DNA sample is digested into fragments by one or more restriction enzymes, and the
resulting restriction fragments are then separated by gel electrophoresis according to their size.
The basic technique for the detection of RFLPs involves fragmenting a sample of DNA with the application of
a restriction enzyme, which can selectively cleave a DNA molecule wherever a short, specific sequence is
recognized in a process known as a restriction digest.
The DNA fragments produced by the digest are then separated by length through a process known as agarose
gel electrophoresis and transferred to a membrane via the Southern blot procedure.
Hybridization of the membrane to a labeled DNA probe then determines the length of the fragments which
are complementary to the probe. A restriction fragment length polymorphism is said to occur when the length
of a detected fragment varies between individuals, indicating non-identical sequence homologies. Each
fragment length is considered an allele, whether it actually contains a coding region or not, and can be used in
subsequent genetic analysis.
The full RFLP process requires probe labeling, DNA fragmentation, electrophoresis, blotting, hybridization,
washing, and autoradiography. The detected RFLP is visualized using X-ray film in autoradiography, where
DNA fragments can be viewed and analyzed after they are separated from one another by electrophoresis.
Analysis of RFLP variation in genomes was formerly a vital tool in genome mapping and genetic disease
analysis. If researchers were trying to initially determine the chromosomal location of a particular disease gene,
they would analyze the DNA of members of a family afflicted by the disease, and look for RFLP alleles that
show a similar pattern of inheritance as that of the disease (see genetic linkage). Once a disease gene was
localized, RFLP analysis of other families could reveal who was at risk for the disease, or who was likely to be
a carrier of the mutant genes. RFLP test is used in identification and differentiation of organisms by analyzing
unique patterns in genome. It is also used in identification of recombination rate in the loci between restriction
sites.
RFLP analysis was also the basis for early methods of genetic fingerprinting, useful in the identification of
samples retrieved from crime scenes, in the determination of paternity, and in the characterization of genetic
diversity or breeding patterns in animal populations.
Applications
Some of the applications for RFLP analysis include:
DNA Fingerprinting: Forensic scientists may use RFLP analysis to identify suspects based on evidence
samples collected at scenes of crimes.
Paternity: RFLP is also used in the determination of paternity or for tracing ancestry.
Genetic Diversity: The technique can be used in studying evolution and migration of wildlife,
studying breeding patterns in animal populations and the detection and diagnosis of certain diseases.

2. AFLP: Amplified Fragment Length Polymorphism

AFLP is a PCR-based tool used in genetics research, DNA fingerprinting, and in the practice of genetic
engineering. Developed in the early 1990s by Keygene,
AFLP-PCR is a highly sensitive method for detecting polymorphisms in DNA. The technique was originally
described by Vos and Zabeau in 1993. In detail, the procedure of this technique is divided into three steps:
 Digestion of total cellular DNA with one or more restriction enzymes and ligation of restriction half-
site specific adaptors to all restriction fragments.
 Selective amplification of some of these fragments with two PCR primers that have corresponding
adaptor and restriction site specific sequences.
 Electrophoretic separation of amplicons on a gel matrix, followed by visualisation of the band pattern.
 The resulting data are not scored as length polymorphisms, but instead as presence-absence
polymorphisms.
Applications:
The AFLP technology has the capability to detect various polymorphisms in different genomic regions
simultaneously. It is also highly sensitive and reproducible.
As a result, AFLP has become widely used for the identification of genetic variation in strains or closely
related species of plants, fungi, animals, and bacteria.
The AFLP technology has been used in criminal and paternity tests, also to determine slight differences within
populations, and in linkage studies to generate maps for quantitative trait locus (QTL) analysis.
Advantages:
There are many advantages to AFLP when compared to other marker technologies including randomly
amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP).
AFLP not only has higher reproducibility, resolution, and sensitivity at the whole genome level compared to
other techniques, but it also has the capability to amplify between 50 and 100 fragments at one time. In
addition, no prior sequence information is needed for amplification.
As a result, AFLP has become extremely beneficial in the study of taxa including bacteria, fungi, and plants,
where much is still unknown about the genomic makeup of various organisms.

3. RAPD: Random amplified polymorphic DNA

Random amplified polymorphic DNA (RAPD) is a PCR based technique for identifying genetic variation.
It involves the use of a single arbitrary primer in a PCR reaction, resulting in the amplification of many
discrete DNA products.
The technique was developed independently by two different laboratories (Williams et. al., 1990; Welsh and
McClelland, 1990) and called as RAPD and AP-PCR (Arbitrary primed PCR) respectively.
This procedure detects nucleotide sequence polymorphisms in a DNA amplification-based assay using only a
single primer of arbitrary nucleotide sequence.
 In this reaction, a single species of primer binds to the genomic DNA at two different sites on opposite
strands of the DNA template.
 If these priming sites are within an amplifiable distance of each other, a discrete DNA product is
produced through thermocylic amplification.
 The polymorphisms between individuals result from sequence differences in one or both of the primer
binding sites, and are visible as the presence or absence of a particular RAPD band. Such
polymorphisms thus behave as dominant genetic markers.
 The RAPD technique is based on the polymerase chain reaction (PCR). A target DNA sequence is
exponentially amplified with the help of arbitrary primers, a thermostable DNA polymerase, dideoxy
nucleotide tri - phosphates, magnesium and reaction buffer.
 The reaction involves repeated cycles, each consisting of a denaturation, a primer annealing and an
elongation step. In the first step the DNA is made single stranded by raising the temperature to 94°C
(denaturation).
 In the second step, lowering of the temperature to about 40 to 65°C results in annealing of the primer to
their target sequences on the template DNA (annealing step). In the third cycle, temperature is chosen
where the activity of the thermostable Taq DNA polymerase is optimal, i.e., usually 720 C.
Applications
RAPDs have been used for many purposes, ranging from studies at the individual level (e.g. genetic identity)
to studies involving closely related species.
RAPDs have also been applied in gene mapping studies to fill gaps not covered by other markers. Variants of
the RAPD technique include Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) which uses longer
arbitrary primers than RAPDs, and DNA Amplification Fingerprinting (DAF) that uses shorter, 5-8 bp primers
to generate a larger number of fragments.