Are humans a type of PCR machine

The following points highlight the top eight types of genetic markers. Some of the types are: 1. Single-Nucleotide Polymorphism (SNP) 2. Restriction Fragment Length Polymor­phism (RFLP) 3. Amplified Restriction Fragment Length Polymorphism (AFLP) 4. Random Amplification of Polymor­phic DNA (RAPD) 5. Variable Number Tandem Repeat (VNTR) 6. Cleaved Amplified Polymorphic Se­quences (CAPS).

Genetic Marker Type # 1.

Single-Nucleotide Polymorphism (SNP):

These are positions in a genome where either of two different nucleotides can occur. Some members of the species have one version of the SNP and some have the other version. For example rs6311 and rs6313 are SNPs in the HTR2A gene on human chromosome 13.

SNPs are the commonest forms of polymor­phism. They can be detected by using specific primers to amplify the alleles of interest. These are used to identify allele specific polymorphisms.

Use and Importance:

Variations in the DNA sequences of humans can affect how humans develop diseases and respond to pathogens, chemicals, drugs, vaccines, and other agents. The greatest importance of SNP in biomedical research is in the job of identification by comparing regions of the genome between people belonging to the same population group (cohorts).

The study of SNPs is also important in crop and livestock breeding program. SNPs do not usu­ally function individually, rather, they work in coordination with other SNPs to manifest a diseased condition.

Advantages:

By tracing SNP it is possible to identify muta­tions due to single base substations which are otherwise non-identifiable by conventional mapping.

Genetic Marker Type # 2.

Restriction Fragment Length Polymor­phism (RFLP):

Restriction fragment length polymorphism, commonly pro­nounced “rif-lip”, special types of SNPs, are gene markers which result in a restriction site being changed. When digested with a restriction endonuclease the loss of the site is revealed because two fragments remain joined together.

Analysis Technique:

The basic technique for detecting RFLPs in­volves fragmenting a sample of DNA by a restriction enzyme, which can recognize and cut DNA wherever a specific short sequence occurs, in a process known as a restriction digest. The resulting DNA fragments are then separated by length by agarose gel electro­phoresis, and transferred to a membrane via the Southern blot procedure.

Hybridization of the membrane to a labelled DNA probe then determines the length of the fragments which are complementary to the probe. A RFLP occurs when the length of a detected fragment varies between individuals. Each fragment length is considered an allele, and can be used in genetic analysis.

Applications:

Analysis of RFLP variation in genomes was 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 analyse 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.

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 carriers of the mutant genes.

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.

Advantages:

RFLP markers are co-dominant. Hence iden­tifications of patterns of genomes in homozy­gous and heterozygous individuals are easier.

Disadvantages:

May require large amounts of DNA samples since distinct RFLP markers require different restriction enzymes.

Genetic Marker Type # 3.

Amplified Restriction Fragment Length Polymorphism (AFLP):

Ampli­fied Fragment Length Polymorphism is a PCR-based tool used in genetics re­search, DNA fingerprinting, and in the practice of genetic engineering. AFLP- PCR is a highly sensitive method for de­tecting polymorphisms in DNA. The technique was originally described by Vos and Zabeau in 1993.

Analysis Technique:

Step 1:

DNA Extraction:

In the first step of AFLP clean and high molecular weight DNA is extracted using CTAB procedure.

Step 2:

Restriction Digestion:

Restriction fragments of the genomic DNA are pro­duced by using two different restric­tion enzymes: a frequent cutter (the four-base restriction enzyme Msel) and a rare cutter (the six-base restriction enzyme EcoRI).

The frequent cutter serves to generate small fragments, which amplify well and which have the optimal size range for separation on a sequence gel, whereas the rare cutter limits the number of fragments to be amplified.

Step 3:

Ligation of Oligonucleotide Adapt­ers:

Double-stranded adapters consist of a core sequence and an enzyme-specific sequence . Therefore, adapters are specific for either the EcoRI site or the Msel site. Usually restriction and li­gation take place in a single reaction.

Ligation of the adapter to the re­stricted DNA alters the restriction site in order to prevent a second restric­tion from taking place after ligation has occurred. The core sequence of the adapters consists of a known DNA se­quence of 20 nucleotides, which will be used later as primer in the PCR.

Step 4:

Pre-Amplification:

This step is a nor­mal PCR where the adapters are used as primers. This first PCR, called pre-amplification, allows a first selec­tion of fragments by only amplifying the DNA restriction fragments that have ligated an adaptor to both ex­tremities.

Step 5:

Amplification:

The aim of this step is to restrict the level of polymorphism and to label the DNA. For this second amplification, we added three more nucleotides at the 3′ end of the primer sequence used for the pre-amplification (adaptors sequence + 3 nucleotides). These two additional nucleotides make the amplification more selective and will decrease the number of restriction fragments amplified (polymorphism).

Step 6:

Electrophoresis:

The PCR products are denaturized and run on acrylamide gel.

Applications:

The AFLP technology has the capability to detect various polymorphisms in different ge­nomic regions simultaneously. AFLP is 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, 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), and microsatellites.

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.

Disadvantage:

They are typically dominant markers which are unable to provide details on homozygous and heterozygous nature of the gene.

Genetic Marker Type # 4.

Random Amplification of Polymor­phic DNA (RAPD):

The RAPD, pro­nounced “rapid”, utilizes short synthetic oligonucleotides of random sequences as primers to amplify Nano gram amounts of total genomic DNA under low annealing temperatures by PCR. Amplification prod­ucts are generally separated on agarose gels and stained with ethidium bromide.

At an appropriate annealing temperature during the thermal cycle, oligonucleotide primers of random sequence bind several priming sites on the complementary se­quences in the template genomic DNA and produce discrete DNA products if these priming sites are within an amplifiable distance of each other (Fig).

The profile of amplified DNA primarily depends on nucleotide sequence homology between the template DNA and oligonucleotide primer at the end of each amplified product. Nucleotide variation between different sets of template DNAs will result in the presence or absence of bands because of changes in the priming sites (Fig. 8.5).

Applications:

1. Used in genetic mapping and DNA finger printing.

2. Sex determination.

3. Generation of specific PCR primers for anonymous Genomes.

4. Quantitative analysis of mixed bio-samples.

5. Determination of paternity and kinship relationships.

6. Analyses of interspecific gene flow and hybrid speciation.

7. Determination of taxonomic identity.

Advantage:

Can use random primer sequences.

Disadvantages:

(1)RAPD markers are dominant. Hence, dis­tinguishing homozygous and heterozygous individuals is not possible.

(2) Reproducibility of the experiment is very low since low annealing temperatures are used.

(3) PCR conditions greatly affect the selective amplification of polymorphic bands.

Genetic Marker Type # 5.

Variable Number Tandem Repeat (VNTR):

A Variable Number Tandem Re­peat (VNTR) is a location in a genome where a short nucleotide sequence is organized as a tandem repeat. These can be found on many chromosomes, and of­ten show variations in length between individuals. Each variant acts as an in­herited allele. Due to this reason VNTR can be used for personal or parental identification.

Use of VNTRs in Genetic Analysis:

VNTRs are frequently used in the develop­ment of linkage maps. Now that many ge­nomes have been sequenced, VNTRs have be­come essential to forensic crime investiga­tions, via DNA fingerprinting. When removed from surrounding DNA by the PCR or RFLP methods, and their size determined by gel electrophoresis or Southern blotting, they produce a pattern of bands unique to each individual.

When tested with a group of inde­pendent VNTR markers, the likelihood of two unrelated individuals having the same allelic pattern is extremely improbable. In the ex­ample considered in the diagram below locus A is a tandem repeat of the motif GC: there are four alleles, with two, three, four, or five repeats (A2, A3, A4, and A5, respectively).

Locus B is a tandem repeat of the motif AGCT: there are only two alleles, with two or three repeats (B2 and B3, respectively). Individual 1 is heterozygous at Locus A (A2/A5) and ho­mozygous at Locus 2 (B2/B2) which gives a single-banded phenotype in the fingerprint.

Individual 2 is heterozygous at both loci (A4/ A3 and B3/B2). The two individuals are dis­tinguishable at either locus. Typical finger­prints include a dozen or more VNTR loci. VNTR analysis is also being used to study ge­netic diversity and breeding patterns in populations of wild or domesticated animals.

Genetic Marker Type # 6.

Cleaved Amplified Polymorphic Se­quences (CAPS):

The principle is simi­lar to RFLP procedure. Here, PCR ampli­fication is used in place of DNA blot hy­bridization. The PCR is used to specifically amplify particular regions of the genomic DNA. Selection is achieved by use of spe­cific primers. The amplified fragments are then digested with restriction endonucleases which reveal the DNA polymor­phism.

Advantages:

(1) CAPS markers are co-dominant in nature allowing identification of homozygous and heterozygous patterns with ease.

(2) Require only smaller amounts of DNA since PCR amplification is employed.

(3) Can be easily assayed with agarose gel electrophoresis.

Disadvantage:

(1) The procedure requires use of specific primers and hence the sequence must al­ready be known.

Genetic Marker Type # 7.

Sequence Tagged Site (STS):

An STS marker is a DNA sequence found once per haploid set of genome which can be ampli­fied by PCR technique. Hence, it is unique to the genome and, therefore, can map ge­nomes efficiently. STS markers can effi­ciently incorporate different information to create a physical map. These markers represent coding regions and are, there­fore, widely used in genetic researches.

Genetic Marker Type # 8.

Simple Sequence Repeats (SSR): Microsatellites:

Microsatellites are short sequences of DNA repeated in the genome. These motifs are tandem repeats and are usually polymorphic in nature because of the variations in the repeat units. These are called Short Sequence Repeats or SSR.

Specific primers complementary to the re­gions of microsatellites are used to iden­tify the SSRs. The hybridization depends on the number of repeats and so does the amplified fragment size. Agarose or polyacrylamide gel electrophoresis is used to separate these polymorphic fragments.

Advantages:

(a) Does not employ restriction enzymes and avoid the problems such as partial diges­tion.

(b) They are co-dominant in nature.