recombinant dna technology

1. 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

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 fromThermus 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+

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. 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 reactionPCR 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. 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