PCR can be used for cloning, DNA fingerprinting, gene expression analysis, DNA sequencing, and more. It works by amplifying a specific region of DNA through repeated heating and cooling cycles. This allows very small amounts of DNA to be exponentially multiplied, enabling various applications. PCR was invented in 1983 by Kary Mullis and revolutionized molecular biology.

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 28th 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
1. Denaturation
 90°C - 100°C
 Denaturation of DNA
2. Annealing
 30°C - 65°C
 Primer binds to both strands, antiparallel
3. Extension
 60°C - 75°C
 Addition of dNTPs at 3prime end

13. Polymerase Chain Reaction

15. 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

16. 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

17. 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.

18. 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

19. 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.

20. 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.

21. PCR Application

22. 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.

23. 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.

24. 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.

25. 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

26. Mutation detection in Inherited disease
 Any point mutation, a deletion or an insertion and expanded tandem tri-
nucleotide 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.

27. 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.

28. 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.

29. 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.

31. 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.

33. 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.

35. 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

36. 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.

38. PLASMID CLONING BY
PCR

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

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

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

42. 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

43. 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.

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

45. 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.

46. 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.

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

48. 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.

49. 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.

50. 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.

51. Mutation Detection
Through PCR cloning

52. 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.

53. 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

54. MLPA (Multiple ligation-dependent probe amplification)
 MLPA used to detect mutations up to 50 DNA nucleotide sequence.
 For addition and deletion mutations.
hybridization of
Probe to
genomic DNA
Probe has 2
parts, 1st half
consists of
specific
sequence and
primer, other
half consists of
random
fragment.
Two probes
hybridize on
target gene and
join with help
of ligase.
PCR helps
amplification of
probe.
Then copy
number of our
target DNA can
be evaluated
and measured.

56. Single stranded conformational polymorphism
 Screening for unknown mutations.
 Comparison between denatured wild type and mutant fragments during gel
electrophoresis.
Denature
fragments of
DNA
Renature
avoiding
formation of
double strands
Primary
nucleotide
sequence
results this
conformation
Polyacrylamide
gels are used
for screening
PCR
amplification
of these
fragments
80-90% point
mutations can
be detected.