Pcr and its applications in cloning

polymerase chain reaction
applications

1. PCR and its Applications in Cloning Presented to: Dr. Sumaira Rasool

2. Before PCR Scientists used two approaches to multiply DNA Using vectors to duplicate DNA molecules Cut DNA with Restriction enzymes run on gel purifying required DNA Multiplying by vectors

3. Discovery PCR was invented by Kary Mullis Born on 28 th December 1944, Kary Mullis is a noble prize winning biochemist, Author and Lecturer Started working at Citus Corporation, California in 1973

4. How PCR was invented? thinking to use simpler method for DNA sequencing Planning to use sanger method called “ dideoxytechnique ” Use dideoxybases (i.e. O removed from 2prime position of carbon ring) & polymerase Act as a cap Stop further addition of bases

5. Sanger's technique

6. Discovery Then he thought of using 2 oligonucleotides & ddNTP , that will bracket the specific base

7. Problem and Solution Problem was, free bases in the solution may also bind to the extending sequence To overcome this problem he planned to use two polymerases, First will bind all free dNTPs Denature these molecules Add second polymerase that will only bind ddNTP But the chances are ddNTP will also bind to extended sequences because they are in the solution

8. At that point he came to realize that the extended sequences are complementary of the original sequence It had no effect on sequencing It will just multiply number of DNA molecules 2, 4, 6, 8,………………., 32,……………………….

9. First PCR He did first experiment with 25bp long DNA Primers of 11 & 13bp 98° C & 60° C cycles of 1-2 min Originally used polymerase enzyme had to be added again in every cycle Taq polymerase bacteria Thermus Aquaticus lives in hot springs Stable at high temperature Only added once at the beginning Mullis, K. (1990). Inicio de la PCR. Scientific American , 56–65.

10. Working of PCR works on consecutive heating and cooling cycles. The basic protocol of a PCR require following ingredients; Template DNA Pair of Primers DNA polymerase (usually Taq Polymerase) dNTPs (i.e. four bases A T G C ) some ions and salts are also added

11. Working of PCR works on consecutive heating and cooling cycles. The basic protocol of a PCR require following ingredients; Template DNA Pair of Primers DNA polymerase (usually Taq Polymerase) dNTPs (i.e. four bases A T G C ) some ions and salts are also added

12. PCR Mechanism Three steps Denaturation 90°C - 100°C Denaturation of DNA Annealing 30°C - 65°C Primer binds to both strands, antiparallel Extension 60°C - 75°C Addition of dNTPs at 3prime end

13. Polymerase Chain Reaction

14. Types There are many different types of polymerase chain reaction on the basis of their principles. Real time PCR Reverse transcriptase PCR Quantitative real time PCR Multiplex PCR Nested PCR

15. 1. Real time PCR Real time PCR is introduced by Higuchi and fellows in 1992. In Real Time PCR we find out an accurate quantification of DNA sequence in a complex mixture. Real Time PCR is based on the fluorescense technology in which dyes are present in fluorescenrt repoter molecules that binds to the double stranded DNA or sequence specific probe. Real Time PCR is divided into two types: Non-specific detection using binding dyes Specific detection target specific probes

16. Non-specific detection using binding dyes In real time PCR, DNA binding dyes are used as fluorescent protein work as reporter molecules. Fluorescent reporter molecules increase as the reaction proceeds. In real time PCR technique only that product is detected which is formed at the exponential phage of reaction. Different dyes are used but SYBR Green is commonly used dye in Real Time PCR. It is the specific double stranded DNA dye. Fluorescense increase when SYBR Green binds to the double stranded DNA. Ethidium bromide is not used as SYBR Green.

17. Specific detection target specific probes Oligonucleotides probe are used for the specific detection . Oligonucleotides are labelled with fluorescent dye. Different types of probes are used for the detection of real time PCR. These include: Taq man probes FRET hybridization probes Molecular beacons

18. 2. Reverse Transcriptase Polymerase Chain Reaction Reverse transcriptase PCR is a modified technique used for the detection of RNA expression. In this technique, RNA is used rather than DNA. In real time PCR, transcriptase enzyme is used for converting mRNA into cDNA . cDNA is converted into double stranded DNA with the help of DNA polymerase enzyme. when primer is attached with cDNA and second strand is complete. DNA strands are now denatured then add two primers for the synthesis of cDNA. When second strand of DNA is formed, this strand is heat denatured.

19. Continued….. Now both strands are synthesizing into new strands when second primer is added. Electrophoresis technique is used to detect the presence of amplified fragment.

20. PCR Application

21. Paternity Testing Genetic material is inherited from both parents, half from mother and half from father. Paternity and maternity test can be performed matching genetic fingerprinting of suspected parents with that of children. DNA sample from buccal saliva or blood is collected and extracted from the alleged child. Then the extracted DNA is subjected to PCR, thousands of copies of amplified DNA is obtained. Once amplification is achieved the DNA sequence of child and parents are matched.

22. Procedure PCR nucleotide sequence of child and suspected father is run on the gel by applying electric current. The nucleotide sequence of PCR product may resemble to either father 1 or 2. If the sequence match this show the relatedness between the child and the parent Resemblance of nucleotide sequences of the two is based on electric charge and molecular size.

23. DNA analysis of archaeological specimens As DNA is relatively stable and remain intact for a long period of time, PCR can help in analysis of DNA from those embedded materials. It plays an important role in phylogenetic analysis. Minute quantities of DNA from any source such a fossilized material, hair, bones, mummified tissues can be amplified using PCR technique.

24. Diagnosis of Infectious Disease PCR technology facilitates the detection of DNA or RNA of pathogenic organisms in clinical diagnostic tests for a range of infectious agents like viruses, bacteria, protozoa etc. DNA from the infected parts of a person or animal may be subjected to PCR with primer specific gene of the pathogen and diagnosis can be done on amplification of DNA. These PCR-based tests have advantage over conventional antibody-based diagnostic methods that determine the body's immune response to a pathogen. As patients can take weeks to develop antibodies against an contagious agent but this is fast and efficient. PCR-based tests have been developed to enumerate the amount of virus in a person's blood (‘viral load') there by allowing physicians to check their patients' disease progression

25. Mutation detection in Inherited disease Any point mutation, a deletion or an insertion and expanded tandem tri­nucleotide repeat can be detected by PCR. Somatic mutations in oncogenes or tumor repressor genes can also be detected by PCR with primers flanking the insertions or dele­tions. It is a highly sensitive tool in the diagnosis of various diseases in human. The occurrence of genetic diseases can be identified by the length of Restriction fragment length polymorphism (RFLP). Incase of genetic diseases, there is a mutation resulting in a detectable change in the length of the restriction fragment.

26. Detecting HIV HIV DNA collected from white blood cells of patient. Using PCR DNA is amplified, with each cycle thousands of copies of DNA are obtained. DNA detection either by radioactive probe or biotinylated probe. After amplification, the DNA is transfer to the nitrocellulose membrane and react with radiolabeled probe. Complementary nucleotide sequence hybridizes Radioactivity is determined.

27. Gene Expression Analysis Testing gene expression can be done by PCR Important enzyme used in this method is reverse transcriptase. Gene expression can be test by using the RNA. The more abundant transcripts from highly transcribed genes (now in the form of cDNA) will yield more product than more weakly transcribed genes.

28. Steps Isolate RNA. Convert total mRNA to cDNA with reverse transcriptase. PCR amplify a gene of interest using total cDNA as template. Primers for this step will flank at least one intron in the gene, if possible. This will allow you to distinguish the desired product from cDNA amplification versus product from amplification of contaminating genomic DNA, a common problem.

29. DNA Fingerprinting PCR's main advantage in forensics is that forensic scientists can use it to amplify or make copies of regions of the genome that vary widely between different individuals, called VNTRs (variable number tandem repeats). By comparing the length of different VNTRs they can determine whether the sample may be a match with the suspect's DNA.  Scientists can use it to amplify VNTRs from the sample even if only trace amounts of DNA are present initially. Often forensic scientists must work with very small amounts of DNA, so the ability to use a small or partially degraded sample is vital.

30. DNA cloning  PCR can be utilized for DNA cloning - It can concentrate fragments for insertion into a vector from a bigger genome, which may be accessible in little amounts A little amount of DNA is necessary . PCR cloning is a rapid method for cloning genes, and is often used for projects that require higher throughput than traditional cloning methods can accommodate. It allows for the cloning of DNA fragments that are not available in large amounts.

31. DNA sequencing The DNA to be sequenced is used in the single stranded form and DNA polymerase synthesizes new complementary DNA strand. Nucleic acids are labeled Detection of cDNA and along the original DNA sequence

32. Purify a gene The polymerase chain reaction can also be used to obtain a pure sample of a gene The region of the starting DNA molecule that is copied during PCR is the segment whose boundaries are marked by the annealing positions of the two oligonucleotide primers. A PCR experiment can be completed in a few hours But it took weeks if not months to obtain a gene by cloning.

33. PLASMID CLONING BY PCR

34. DEFINITION It is a method of copying piece of DNA by adding restriction sites to ends of DNA to clone into plasmid easily.

35. IMPORTANCE For cloning genes required in high amount Not accomplished by traditional cloning Helpful in getting large amounts of DNA

36. RUN PCR & PURIFY PCR PRODUCT Amplify DNA by PCR Highly efficient TAQ Polymerase used Annealing temp = Melting temp For plasmid use multiple annealing temp to reduce mis -priming. Use PCR purifying kit to isolate PCR product. Product ready for PCR.

37. DIGESTION OF DNA Restriction digest PCR product & recipient plasmid. Occur 24 hrs. or overnight. If we need no overhangs then use phosphatase. E.g. shrimp or calf alkaline phosphatase

38. ISOLATION BY GEL ELECTROPHORESIS Performed for isolation of DNA. Use wide combs. Run gel slowly. Space lanes between samples. Importance: to visualize PCR product & check band efficiency.

39. LIGATION Insert sequence inside plasmid. 100ng of DNA recommended. Using Taq polymerase takes 15 min.

40. TRANSFORMATION Transform 2uL ligation reaction into competent cell media. No. of bacterial colonies tells success of transformation. For high no. of colonies there is no need of ligase to ligate plasmid. If we add ligase and have more colonies, it will be a result of self-ligated plasmid.

41. Isolate the plasmid To know success rate of ligation check Bacterial colonies. To check purification take 10 colonies & grow overnight. Run digestion on gel, we see 2 bands representing size of vector & insert.

42. VERIFICATION Chances of mutation can occur depends on polymerase used. Sequencing can be done.

43. APPLICATIONS Plasmid can play a role in the field of gene therapy, they are used for insertion of therapeutic genes to fight against diseases. Plasmid can be used in recombinant DNA technology to add desired drug. Expression of genes of interest. There replication in bacteria is easy. They can be easily manipulated.

44. APPLICATIONS Determine variations in nucleotide bases (SNPs). Enabled study of gene function. Helpful in preventing and treating diseases. Antibiotic resistance can be pass in to the body to prevent growth of harmful bacteria.

45. ADVANTAGES A nanogram of DNA is enough for amplification. No need for re-isolation or enzymes. Error possibility is less. No need for intensive labors.

46. Mutation Detection Through PCR cloning

47. Mutation Detection There are many techniques available to identify mutations. With PCR cloning it became easier to tell whereabouts of mutations. After amplification with PCR, DNA microarray can also be used for testing of multiple mutations. Example Small sample of DNA containing β -globin gene can be amplified to check if the person have sickle cell anemia.

48. Mutation Detection PCR cloning can be used to check various disorders from a single human cell. Once it was used to screen in-vitro fertilized human embryo whose parents were both carriers of cystic fibrosis. With utilization of PCR cloning career couples can be guaranteed of having a baby who wont have cystic fibrosis. PCR cloning can also help in early discovery of HIV

49. MLPA (Multiple ligation-dependent probe amplification) MLPA used to detect mutations up to 50 DNA nucleotide sequence. For addition and deletion mutations.

50. Single stranded conformational polymorphism Screening for unknown mutations. Comparison between denatured wild type and mutant fragments during gel electrophoresis.