This document discusses several biotechnological tools used for diagnostics: 1. DNA isolation, restriction enzyme digestion, polymerase chain reaction (PCR), gel electrophoresis, and DNA sequencing. PCR is described as artificially multiplying genetic material using primers and DNA polymerase. DNA sequencing methods like Sanger sequencing and Maxam-Gilbert are also outlined. The document concludes by briefly discussing gene cloning techniques like recombinant DNA technology and PCR for preparing copies of DNA fragments.

1. Biotechnological tools used
for diagnosticS
Dr. Sunita
Dr. Mrinalini Saran

3. 1.DNA ISOLATION

5. 2.Restriction enzyme digest

13. 3.POLYMERASE CHAIN REACTION
(PCR)

14. PCR:-
 A process used to artificially multiply a chosen piece of
genetic material.
 May also be known as DNA amplification.
 One stand of DNA may yield 230 strands or more.

16. Uses of PCR-
 DNA sequencing
 Gene cloning
 DNA profiling
 Transformation
 Making artificial genes

17. DNA selection
 DNA is selected either as a complete chromosome or a
fragment.
 Primers are constructed that will bind within a desired
region(purple).
 Additional reaction chemicals are added.

18. The reaction mixture
 Water and pH buffer.
 DNA to be multiplied
 RNA Primers
 Nucleotides
 DNA Polymerase (Taq)

20. Splitting DNA
 DNA is heated to 950c which causes double
stranded DNA to become single stranded.

21. Adding Primers
 RNA primers are prepared that base pair with a
selected sequence of DNA.
 Two primers must be used. One for each strand of
DNA.
 The reaction temperature is lowered to 600c to
allow the primer to attach(anneal).

22. Adding nucleotides
 The reaction temperature is raised to 720c to allow
nucleotides to be added.
 Polymerase enzyme (Taq) catalyze the addition of
nucleotides.
 Nucleotides are added in a 5´ to 3´ direction.

23. Repeating the cycle
 In the next cycle the heating and cooling steps are
repeated.
 The original (red/purple) strands reproduce as per the
first cycle.
 The new strands only duplicated between the primer
site to produce blocks of a set length.

25. Continuing the cycle
 The cycle is then repeated over and over again.
 With each cycle the no. of short fragments rapidly
increases while the no. of larger fragments increases
slowly.

26. 30 cycles
 After 30 cycles, if replication has occurred fully, a total
of 2,147,483,648 strands could be produced.
 All but 62 will be the short length of the desired DNA
fragment.

28. 4.Gel electrophoresis

39. 5.DNA Sequencing

40. DNA SEQENCING
 Determining the order of bases in a section of DNA.
 To analyze gene structure and its relation to gene
expression as well as protein conformation.

41. Purpose
 Deciphering “ code of life”.
 Detecting mutation.
 Typing microorganisms.
 Identifying human halotypes .
 Designating polymorphisms.

42. Maxam and Gilbert method
 A.M.Maxam and W.Gilbert-1977
 The sequencing of a double stranded or single strand
DNA molecule is determine by treatment with
chemicals that cut the molecule at specific nucleotide
position.

43. Sanger method termed as chain
termination method
 This method uses dideoxynucleotide triphosphates
(dint's) chain termination :
Which have an H on the 3’
carbon of the ribose sugar instead of the normal OH
found in deoxynucleotide triphosphates(dNTPs).
There for in a synthesis reaction , if a
dideoxynucleotide is added instead of the normal
deoxynucleotide , the synthesis stops at that point
because the 3’OH necessary for the addition of the
next nucleotide is absent.

44. comparison
1 Sanger method
 Enzymatic
 Requires DNA synthesis
 Termination of chain
elongation
 Automation
 Single-stranded DNA
2 Maxam Gilbert Method
 Chemical
 Requires DNA
 Break DNA at different
nucleotides
 Automation is not available
 Double-stranded or single-
stranded DNA

45. Applications of DNA sequencing
 Forensics: to help identify individual has a different
genetic sequence.
 Medicine: can be used to help to detect the genes
which are linked to various genetic disorders such as
muscular dystrophy.
Agricultural : the mapping and sequencing of a
genome of microorganisms has helped to make them
useful for crops and food plants.

46. Advantage:-
 Improve the health care.
 Helping plants and animals to be able to resist certain
disease.
 Helps in forensic science for identifying criminals.

47. Disadvantage:-
 The risk that the DNA sequencing may not work right
and provide the wrong DNA sequencing of the
fragment.

48. 6. Gene cloning

50. DNA Cloning Techniques
 Technique for gene cloning enable scientists to prepare
multiple copies of DNA pieces.
 DNA pieces are stored in DNA libraries for easy
identification and accessibility.
 DNA cloning can occur (in vitro) via RECOMBINANT
DNA TECHNOLOGY and POLYMERASE CHAIN
REACTION (PCR).

51. MATERIALS AND METHODS
 Reagents-1) Taq enzyme
2)10X buffer Teq w/o Mgcl₂
 Restriction enzyme -1) Xbal and Hind III
2)T4 DNA ligase
 Scarlett Barley Malt was provided by Zhong liang Malt
Division
 Wheat Malt obtained from Weyermann .

53. THANK
YOU