Pcr

recombinant dna technology

1. Molecular techniques PCR, RFLP AND RAPD

2. common methods used in molecular biology biochemistry genetics and biophysics involve manipulation and analysis of DNA,RNA, Protein and lipid

3. P olymerase chain reaction

4. Polymerase chain reaction Developed in 1983 by  Kary Mullis Its an invitro technique It is a technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Relies on thermal cycling Older PCR Automated PCR

5. History In 1983, Kary Mullis, PhD, a scientist at the Cetus Corporation, conceived of PCR as a method to copy DNA and synthesize large amounts of a specific target DNA In 1986, Cetus scientists isolated the Taq polymerase from Thermus aquaticus , a bacterium found in hot springs. In 1987, PerkinElmer, another US-based biotech company, launched a thermal cycler, an instrument that is programmed to regulate the temperature of a reaction, heating or cooling the samples as needed. In 1991, Roche bought the rights to PCR from Cetus and invested in refining the science for use in molecular diagnostics to detect diseases. In 1993, Dr, Kary Mullis shares Nobel prize in chemistry for conceiving PCR technology.

6. principle PCR is based on the mechanism of DNA replication in vivo: dsDNA is unwound to ssDNA , duplicated, and rewound. This technique consists of repetitive cycles of: • Denaturation of the DNA through melting at elevated temperature to convert double-stranded DNA to single-stranded DNA • Annealing ( hybridisation ) of two oligonucleotides used as primers to the target DNA • Extension of the DNA chain by nucleotide addition from the primers using DNA polymerase as catalyst in the presence of Mg2+ ions.

7. requirements Target DNA Atleast one intact copy of target gene is required. Increase in copies increases the probability of successful DNA amplification size < 0.1 to few kilobases amount of DNA used is 0.05 to 1.0µg free from heparin, heme , formalin, Mg2+ and detergents Primers length 16 to 30 nucleotides avoid stretches of polybase sequences, repeating motifs and inverted sequences 3' end of the primer should be rich in G, C bases concentration of oligonucleotides must be upto 1µM

8. Continued…. DNA polymerase The first thermostable DNA polymerase used was the Taq DNA polymerase isolated from the bacterium Thermus aquaticus .

9. Continued…. Reaction buffers and MgCl2 Buffer composition depends on the type and characteristics of the enzyme being used and most suppliers usually provide a 10x buffer for use with the respective enzyme. The most common reaction buffer used with Taq / AmpliTaq ® DNA polymerase contains: • 10 mM Tris, pH 8.3 • 50 mM KCl • 1.5-2.5 mM MgCl2 Mg2+ ions: • form a soluble complex with dNTPs which is essential for dNTP incorporation, • stimulate polymerase activity, • increase the Tm of primer/template interaction (and therefore they stabilise the duplex interaction).

10. Deoxyribonucleoside triphosphates Free deoxyribonucleoside triphosphates ( dNTPs ) are required for DNA synthesis. The dNTPs concentrations for PCR should be 20 to 200 μM for each dNTP The four dNTPs should be used at equivalent concentrations to minimize misincorporation errors . pH 7.0-7.5 Continued….

11. Procedure Initialization : 94 to 96 celsius for 1 to 9 mins Denaturation : 94 to 98 celsius for 20 to 30 seconds Annealing : 50 to 65 celsius for 20 to 40 seconds Extension : 75 to80 celsius for 1 min Final Extension : 70 to 74 celsius for 5 to 15 mins Hold : 4 to 15 celsius for short term storage

12. Advantages of PCR Because of its simplicity, PCR is a popular technique with a wide range of application which depend on essentially three major advantages of the method: Speed and ease of use Sensitivity Robustness

13. Variations Allele-specific PCR : a diagnostic or cloning technique based on single-nucleotide variation (single-base differences in a patient). Asymmetric PCR : preferentially amplifies one DNA strand in a double-stranded DNA template. It is used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required. Dial-out PCR : a highly parallel method for retrieving accurate DNA molecules for gene synthesis. Digital PCR ( dPCR ) : used to measure the quantity of a target DNA sequence in a DNA sample. Inverse PCR : is commonly used to amplify DNA sequences which are away from the primers. Ligation-mediated PCR : uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers

14. Nested PCR : increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs. Quantitative PCR ( qPCR ) : used to measure the quantity of a target sequence (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA , or RNA.  Reverse Transcription PCR (RT-PCR) : for amplifying DNA from RNA.Reverse transcriptase reverse transcribes RNA  into cDNA , which is then amplified by -PCR is widely used in expression profiling, Multiplex-PCR : consists of multiple primer sets within a single PCR mixture to produce  amplicons  of varying sizes that are specific to different DNA sequences.. Nanoparticle -Assisted PCR ( nanoPCR ) : In recent years, it has been reported that some nano particles (NPs) can enhance the efficiency of PCR (thus being called nanoPCR ), Continued….

15. applications Medical application like genetic testing, tissue typing , mutation study etc., Infectious disease diagnosis like tests for HIV and Tuberculosis Forensic applications like genetic fingerprinting, Parental testing Research works like selective DNA isolation , Amplification and Quantification of DNA

16. Random Amplified Polymorphic DNA

17. Random amplified polymorphic DNA (RAPD) is a PCR based technique for identifying genetic variation. It involves the use of arbitrary primer in a PCR reaction, resulting in the amplification of many discrete DNA products. It is a lab technique used to amplify unknown (random) DNA segments The randomly amplified polymorphic DNA’s are used as molecular markers because these are polymorphic in nature Introduction

18. Gene A Gene B Gene C This DNA fragment contains 3 genes. A scientist is interested in amplifying only gene B The scientist prepares 2 primers which will anneal to each end of gene B PCR reaction Only gene B is amplified and can then be purified for further analysis RAPD Analysis

19. RAPD Analysis 1 RAPD Analysis 2 1 2 3 4 5 6 Product A Product B 4 5 6 1 2 Product B PCR reaction PCR reaction

20. procedure The DNA of selected species is isolated An excess of selected decaoligonucleotide added This mixture is kept in a PCR equipment and is subjected to repeated cycles of DNA denaturaion – renaturation – DNA replicaation During this process, the decaoligonucleotides will pair with the homologus sequence present at different locations in DNA DNA replication extend the decaoligonucleotide and copy the sequence continuous with the sequence with which the selected oligonucleotides has paired The repeated cycles of denaturation – renaturation – DNA replication will amplify this sequence of DNA

21. Amplication will takes place only at those regions of genome that has the sequence complementary to decaoligonucleotide at their both ends. After several cycles of amplification the DNA is subjected to gel elctrophoresis The amplified Dna will form a distinct band. It is detected by ethidium bromide staining and visible Continued…

22. Advantages it can be used with uncharacterized genomes and can be applied to cases in which only small quantities of Dna are available it can be used on any Dna sample it is an inexpensive yet powerful tool for typing of bacterial species Can be used to study genetic polymorphism between closely related species Can be used to select variants of microbial isolates Limitations the primer must be right for right result it has low power resolution requires large genome template quality of DNA affects the outcome

23. R estriction fragment Length polymorphism

24. Restriction fragment length polymorphism denotes that single restriction enzymes produces fragment of different lengths from the same stretch of genomic DNA of different strains or related species It refers to a difference between samples of homologous DNA molecules from differing locations of restriction enzyme sites, and to a related laboratory technique by which these segments can be illustrated. RFLP analysis was the first DNA profiling technique inexpensive enough to see widespread application. RFLP analysis was an important tool in genome mapping, localization of genes for genetic disorders, determination of risk for disease, and paternity testing. Introduction

25. Procedure Restriction digestion Agarose gel electrophoresis Hybridization Detection by probe labeling Schematic for RFLP by cleavage site loss. Analysis and inheritance of allelic RFLP fragments1 Schematic for RFLP by VNTR length variation.

26. Applications Genome mapping Genetic fingerprinting Genetic disease analysis Characterization of genetic diversity or breeding patterns in animal population

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