**1. Introduction**

Domesticated Sheep (*Ovis aries*) are a major source of meat, wool, milk, and pelts/skin or fur, around the s the generic name of hair from animals such as goat, camel, vicuna, alpaca, angora rabbit and yak [1]. Wool grows from follicles on the sheep's skin, similar to the way that hair grows on human skin. Wool fibres are resistant to sunlight, ultraviolet radiation, heat and fire [2]. Because of the unique attributes of wool (excellent insulative properties, breathability and fire resistance [2], the fibre is widely used for clothing, bedding, carpets and other interior textiles.

High quality pelts are produced from Swakara sheep of Namibia, although there are other pelt-producing sheep breeds. Swakara is a fat-tailed sheep that is very hardy and well adapted to arid conditions and mainly kept for the production of pelts [3–6], though it may also be reared for meat and wool. Originating from Uzbekistan in Central Asia, and imported into Namibia in 1907 (as Karakul sheep then) [3], the intensive research and strategic breeding programmes which were subjected to Karakul sheep in Namibia has resulted in the production of a unique breed named Swakara in Namibia [6]. Swakara pelt production is an exclusive industry that produces outstanding quality pelts characterised by short hair, exceptional patterns and better hair texture [7]. The main product produced from Swakara pelt is high quality leather apparel of various colours that is sought after in the fashion industry. Other accessories, such as hand bags, carpets, shoes, car seat covers, belts are processed.

Wool fibre is made up of three main structures: the cortex, cuticle, and in some coarse wools, the medulla [8]. The cortical cells comprise 90% of the wool fibre, and are responsible for the major physical properties of wool fibre [9]. The cortex consists of the microfibrils, made up of keratin intermediate-filament proteins (KRTs) and embedded in a matrix of keratin intermediate-filament-associated proteins (KAPs) [8, 10, 11] through disulphide cross-linkages [12, 13]. The matrix consists of KAPs, and is divided into three groups based on their amino acid compositions; high-sulphur (HS), ultra-high sulphur (UHS) and high-glycine-tyrosine (HGT) KAPs [8, 14].

The HS KAPs occur at relatively high concentrations in the paracortex when compared to the orthocortex [15], and are encoded by five multigenes families referred to as KRTAP1.n, KRTAP2.n, KRTAP3.n, KRTAP11.n and KRTAP13.n [12]. The HS KAPs are highly conserved at both the amino acid and nucleotide sequence levels [16]. However, there is a consecutively repeated decapeptide unit (QTSCCQPTSI), which varies in its frequency between the HS KAPs [17]. This decapeptide occurs between four times in the KRTAP1.1, three times in the KRTAP1.2, twice in the KRTAP1.3 and five times in the KRTAP1.4 protein [18]. The KAP1.n family is composed of four known proteins, referred to as KAP1.1, KAP1.2, KAP1.3 and KAP1.4, previously referred to as B2A, B2B, B2C and B2D, respectively. The genes that code for the proteins making up the KAPl.n family lack introns and they usually occur in gene clusters [19]. The majority of genes coding for the KAPs have a conserved 18-bp sequence that varies slightly, immediately 5′ to the initiation codon [20, 21]. This suggests that KAPs evolved from a common ancestor, and that their expression may have elements in common.

The KRTAP1.1 gene has been mapped to ovine chromosome 11, clustering with other KRTAP genes [22]. KRTAP1.1 has been reported to be polymorphic in Romney sheep breed of New Zealand [18, 23]; Merino sheep breed of New Zealand [24, 25] and Swakara sheep breed of Namibia [26].

Furthermore, the region spanning the KRTAP1.1/KRTAP1.3/KRT33A loci on ovine chromosome 11 has been associated with variation in wool staple strength in Romney sheep [27].

A number of typing methods have been developed to detect polymorphism in genes that code for traits of economic importance in sheep. These methods are used depending on the specific gene being studied, and the resources available to researchers. In this chapter, the PCR – Applied Fragment Length Polymorphism (AFLP) typing method used to detect polymorphism in the KRTAP1.1 gene is reviewed. Other techniques used to identify polymorphism in keratin genes include PCR - Single strand conformational polymorphism (PCR-SSCP) and PCR - Restriction Fragment Length Polymorphism (PCR-RFLP).

Many studies have described genetic variation within genes that code for the KRTs and the KAPs using PCR-SSCP, including those of Gong *et al*., [28–31], Rogers *et al*., [18], Itenge-Mweza *et al*., [24], Chai *et al.,* [32].

**187**

*Application of PCR Technique to Detect Polymorphism of the KRTAP1.1 Gene in Three Sheep…*

**2. Polymerase chain reaction (PCR)-agarose gel electrophoresis**

reaction mixture [40]. Up to 96 samples can be processed at a time.

loaded into pre-cast wells in the gel and a current applied.

Gel electrophoresis is a technique by which charged molecules are separated according to their size, by moving through a gel while an electric current is being applied [41]. Agarose is a macromolecular substance that is derived from the cell walls of a number of genera of red algae, such as *Gelidium* and *Gracilaria* [42]. It can be purified to a whitish granular powder which, when mixed with water and heated, it sets like a jelly. This is called a gel and it is used as a molecular sieve for the DNA molecules that can be characterised by both charge and size [41]. DNA has a negative charge due to the negative charge of its phosphate groups attached to the 5′ carbon of one nucleotide and the 3′ carbon of the next nucleotide. When put in solution and an electric field is applied, DNA fragments move from the negative (black) terminal to the positive (red) terminal because of the net negative charge in solution [41]. The movement of charged molecules is called migration. DNA is

Developed by Kary Mullis in 1983, Polymerase Chain Reaction (PCR) is a molecular biology technique that is used to produce relatively large numbers of DNA molecules from very small quantity or poor quality. The reaction involves the amplification of a specific segment of the template DNA. A very important requirement is that the sequence of nucleotides on either side of the sequence of interest must be known, so that primers on either side of the sequence of interest can be designed [40]. Primers are short, single-stranded DNA sequence, typically about 18–30 nucleotides in length, that are used as a starting point in DNA synthesis and define the region of the DNA to be amplified. Primers are also referred to as oligonucleotides. In addition to the template DNA and primers, other ingredients needed for the PCR reactions are the DNA polymerase, all four deoxyribonucleotides (dNTP) and magnesium ion (Mg2+). The DNA polymerase used is usually *Taq* Polymerase, isolated from hot springs bacterium, *Thermus aquaticus*, which can withstand the denaturing temperatures [40]. The cycling can be continued without interruption in PCR machines that are simply programmable water baths that accurately and rapidly can change the water temperature that surrounds the

Similarly, many studies have described genetic variation within genes that code for the KRTs and the KAPs using PCR-RFLP. The RFLP patterns were obtained by cutting the KAP1.3 locus defining a 598 bp amplimer using *Bsr* I restriction enzyme in sheep breeds by Xu *et al.,* [33], Chen *et al.,* [9], Kumar *et al*., [34], Mahajan *et al*., [35] and Meena *et al.,* [36]. Parsons *et al*., [37] reported a diallelic polymorphism at the KAP6 locus using *Bam*HI PCR-RFLP to give alleles designated A1 (24.5 kb) and A2 (14.1 kb). Rogers *et al*., [38] reported a di-allelic polymorphism at the KRT33A, formerly known as KRT1.2 defining a 480 bp amplimer in Romney sheep, while Arora *et al.,* [39] found three genotypes (MM, MN, NN) at the KRT33A locus defining 480 bp amplimer in Indian native sheep breeds. Furthermore, Kumar *et al.,* [34] reported three KRT33A genotypes (MM, MN, NN) in Patanwadi and Nali sheep breeds. McLaren *et al*., [22] reported two alleles at the KRT83, formerly known as

*DOI: http://dx.doi.org/10.5772/intechopen.96941*

KRT2.10 locus using a *Bsr* DI.

**2.1 Polymerase chain reaction**

**2.2 Agarose gel electrophoresis**

*Application of PCR Technique to Detect Polymorphism of the KRTAP1.1 Gene in Three Sheep… DOI: http://dx.doi.org/10.5772/intechopen.96941*

Similarly, many studies have described genetic variation within genes that code for the KRTs and the KAPs using PCR-RFLP. The RFLP patterns were obtained by cutting the KAP1.3 locus defining a 598 bp amplimer using *Bsr* I restriction enzyme in sheep breeds by Xu *et al.,* [33], Chen *et al.,* [9], Kumar *et al*., [34], Mahajan *et al*., [35] and Meena *et al.,* [36]. Parsons *et al*., [37] reported a diallelic polymorphism at the KAP6 locus using *Bam*HI PCR-RFLP to give alleles designated A1 (24.5 kb) and A2 (14.1 kb). Rogers *et al*., [38] reported a di-allelic polymorphism at the KRT33A, formerly known as KRT1.2 defining a 480 bp amplimer in Romney sheep, while Arora *et al.,* [39] found three genotypes (MM, MN, NN) at the KRT33A locus defining 480 bp amplimer in Indian native sheep breeds. Furthermore, Kumar *et al.,* [34] reported three KRT33A genotypes (MM, MN, NN) in Patanwadi and Nali sheep breeds. McLaren *et al*., [22] reported two alleles at the KRT83, formerly known as KRT2.10 locus using a *Bsr* DI.
