PCR based molecular markers

Polymerase Chain Reaction ; Types of PCR ; RAPD, ISSR, SSR, AFLP and SCAR markers

1. PCR BASED MOLECULAR MARKERS Presented by DIVYA S I Ph.D. (GPB) 2019608004

2. Polymerase Chain Reaction Karry Mullis - 1983 - Discovered PCR PCR procedure produces microgram ( μg ) quantities of DNA copies (up to billion copies) from even a single copy of the desired DNA. The DNA segment amplified by PCR is often referred to as amplicon . The PCR process has been completely automated and compact thermal cyclers are commercially available.

3. Procedure for PCR Reagents: A target DNA A thermostable DNA polymerase A pair of ~20 nt long oligodeoxynucleotide primers that are complementary to the target DNA fragment The four deoxynucleotide triphosphates - dATP , dCTP , dGTP , and dTTP

4. Denaturation The reaction mixture is first heated most often to 94 degree Celsius to ensure denaturation of the template DNA. The duration of the denaturation step is usually 2 min in the first PCR cycle, but it is only 1 min in the subsequent cycles.

5. Annealing The above mixture is then cooled to a temperature that would allow the primers to anneal to their complementary sequences located at the 3’ ends of the target DNA segment, i.e., the template DNA. Generally the annealing temperature is between 40 and 60 degree Celsius , and the duration of this step is 1 min .

6. Extension In the third and final step, the primers are extended due to the progressive addition of nucleotides to the free 3’-OH groups of the primers and, subsequently, the new strands being synthesized. The temperature during primer extension step is generally maintained at 72 degree Celsius, and the duration of this step is usually 2 min. Taq DNA polymerase is generally able to amplify DNA segments of up to 2 kb.

7. Separation of PCR Amplification Products DNA fragments/amplicons generated by PCR can be separated by electrophoresis in agarose or acrylamide gels . An agarose gel of about 1 % can separate fragment of ~300–1,500 bp. Polyacrylamide gels contain a much more uniform pore size than agarose gels and allow separation of DNA fragments with a higher resolution. A gel containing 6 % acrylamide has a fine network formed by polyacrylamide and can separate DNA fragments differing in length by even one or two base pairs. Polyacrylamide gels are suitable for detection of SSR, AFLP, DNA amplification fingerprinting (DAF), and sequence-tagged sites (STS) markers , while agarose gels are well suited for RFLP and RAPD markers.

8. Types of PCR Reverse Transcription PCR -The enzyme reverse transcriptase is used along with DNA polymerase (reverse transcription PCR) to generate DNA copies of RNA. Realtime reverse transcription PCR - used to estimate the initial quantity of the template RNA. Inverse PCR for amplification of sequences flanking the target sequence. Anchored PCR use for amplification of a target segment when the sequence of only one of its ends is known.

9. Multiplex PCR For amplification of different segments from the same DNA sample different primers may be required and these different primers are added into a single PCR tube. In such cases, a separate PCR reaction will have to be set up for every primer pair because of the difficulties in correct identification of their PCR products. For that different primers may be labeled with different fluorophores, and their PCR products can be distinguished on the basis of color differences in their fluorescence emissions. The PCR products from different primers can be reliably separated by gel electrophoresis if their lengths do not overlap.

10. Advantages and Limitations of PCR It can amplify even a single copy of the target sequence present in a DNA sample and generate millions of copies of this sequence. Further, even partially degraded DNA can be successfully used for PCR. Sequence information for the two ends of the target segment must be known for designing of the primers. Segments of only up to 3 kb are amplified The PCR procedure can often generate “hybrid amplicons” and primer dimers, and it may produce erroneous results due to contaminating DNA. Primer dimers are frequently produced when the two PCR primers have partially complementary 3’ termini.

11. RAPD (Random Amplified Polymorphic DNA) PCR based marker A single short (10 nt long) oligonucleotide with an arbitrary base sequence – primer (forward and reverse primer) Segments of DNA are amplified at random No knowledge of DNA Sequence is required

12. Criteria for amplification

13. RAPD Technique Isolation of DNA Keep the tubes in PCR Thermocycler Denature the DNA DNA strands separated Annealing of primer (36°C) Primer annealed to template DNA strands

14. DNA Synthesis Complementary strand synthesis 35 to 45 cycles Amplified products separated by gel electrophoresis Band detected by Ethidium bromide staining

15. DNA Amplification Fingerprinting DNA amplification fingerprinting amplifies genomic sequences using a single short oligonucleotide, typically, of 4–6 nt as primer , but primers of up to 15 bases can be used. This produces a range of up to 100 short amplified products of different lengths. DAF uses less stringent conditions for annealing and primer extension reactions than PCR. Temperature variation in the thermocycler block is not as crucial in the case of DAF as it is with conventional PCR. Short extension times are sufficient for complete extension of the short products typically obtained in DAF

16. Arbitrary-Primed PCR In arbitrary-primed PCR, arbitrary sequence primers of 18–32 nt are used for amplification. Therefore, amplification can occur only when the annealing conditions allow primer–template pairing with mismatches at some base pairs. In this way, up to 100 bands may be generated for each individual, which are separated by PAGE, and scored as “present”/ ”absent.” The approach is suitable for DNA fingerprinting.

17. RAPD 10 nt length primer More number of amplified fragments (more number of bands) Agarose gel used AP- PCR 18-32 nt length primer Less number of amplified fragments(less number of bands) Polyacyralmide gel used

18. SCAR (Sequenced Characterised Amplified Region Marker) Polymorphic RAPD marker band is isolated from the gel Amplified in the PCR reaction PCR product is cloned and sequenced New longer and specific primer pair of primers (usually, 20–24 nt long) one forward and one reverse primer, specific for the two terminal sequences is designed This primer pair is expected to amplify a single fragment and detect the polymorphism

19. AFLP Procedure In the first step of AFLP procedure, sample genomic DNA is digested with two restriction enzymes One of these enzymes is a rare cutter e.g., EcoRI and the second enzyme is a frequent cutter, e.g., MseI After ligation of adapters to the DNA fragments, their PCR amplification is done in two steps 1) pre- amplification 2) selective amplification

20. SSR (Simple Sequence Repeats) SSRs consist of tandemly repeated sequences of 1–6 bp, of which the dinucleotide repeats (CA)n, (GA)n, and (AT)n are the most frequent and highly polymorphic in eukaryotic genomes. In case of plants, (AT)n and (GA)n repeats appear to be more numerous, while (CA)n repeats constitute one of the most abundant microsatellites in mammals.

21. The simple sequence repeat (SSR) markers are codominant markers in which a microsatellite locus is amplified using a specific primer pair derived from the unique sequences flanking the SSR locus. The unique sequences flanking the SSR loci seem to be conserved within species and even across species within a given genus, but rarely across related genera. Therefore, SSR primers designed on the basis of genome sequence information from one species can be used in a related species as well.

22. Contd., Microsatellites can be amplified for identification by the polymerase chain reaction (PCR) process, using the unique sequences of flanking regions as primers DNA is repeatedly denatured at a high temperature to separate the double strand, then cooled to allow annealing of primers and the extension of nucleotide sequences through the microsatellite. This process results in production of enough DNA to be visible on agarose or polyacrylamide gels. With the abundance of PCR technology, primers that flank microsatellite loci are simple and quick to use, but the development of correctly functioning primers is often a tedious and costly process

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