Kary Banks is considered the great mind behind PCR. He developed PCR in 1985 while working at Cetus Corporation and was awarded the Nobel Prize in 1993. PCR allows for targeted amplification of specific DNA sequences, enabling their analysis even from very small samples. It involves heating and cooling of the DNA sample in the presence of primers, DNA polymerase, and nucleotides to exponentially amplify the target sequence. The amplified DNA can then be analyzed by gel electrophoresis.

2. HistoryHistory
 Great mind behind this PCR: Kary BanksGreat mind behind this PCR: Kary Banks
 MullisMullis
 >Developed PCR in 1985 and was awarded>Developed PCR in 1985 and was awarded
noble prize in 1993noble prize in 1993
 1985- PCR was introduced to the scientific1985- PCR was introduced to the scientific
community at a conference in October.community at a conference in October.

3. PCRPCR
 It targets and amplifies a special region of DNA strand.It targets and amplifies a special region of DNA strand.
 It is an invitro technique to generate large quantities of specifiedIt is an invitro technique to generate large quantities of specified
DNADNA
 It is based on the natural process of DNA replication.It is based on the natural process of DNA replication.
 Two methods currently exist for amplifying the DNA or makingTwo methods currently exist for amplifying the DNA or making
copiescopies
 *Cloning:Takes a long time for enough clones to reach maturity*Cloning:Takes a long time for enough clones to reach maturity
 PCR:Works on even a single molecule quicklyPCR:Works on even a single molecule quickly

4. PCRPCR
 PCR (polymerase chain reaction) is a method toPCR (polymerase chain reaction) is a method to
analyze a short sequence of DNA (or RNA)analyze a short sequence of DNA (or RNA)
even in samples containing only minuteeven in samples containing only minute
quantities of DNA or RNA.quantities of DNA or RNA.
 PCR is used to reproduce (amplify) selectedPCR is used to reproduce (amplify) selected
sections of DNA or RNAsections of DNA or RNA

5. Requirements of PCRRequirements of PCR
 DNA template( contains the sequence of DNADNA template( contains the sequence of DNA
you wanted to amplify. The DNA can be fromyou wanted to amplify. The DNA can be from
animals, plants, viruses, or bacteria. )animals, plants, viruses, or bacteria. )
 PrimersPrimers
 Taq polymeraseTaq polymerase
 Deoxyribonuleoside triphosphates (dNTPs)Deoxyribonuleoside triphosphates (dNTPs)
 Buffer solution is a salt-solution that helps toBuffer solution is a salt-solution that helps to
stabilize the DNA and other components of thestabilize the DNA and other components of the
reaction.reaction.

6. Steps involvedSteps involved
DenaturationDenaturation
 The reaction mixture is heated to a temperatureThe reaction mixture is heated to a temperature
between 90-98 Cbetween 90-98 C
 This breaks the weak hydrogen bonds that holdThis breaks the weak hydrogen bonds that hold
DNA strands together in a helix, allowing theDNA strands together in a helix, allowing the
strands to separate creating single strandedstrands to separate creating single stranded
DNADNA
 Duration of this step is 1-2 minsDuration of this step is 1-2 mins

7. AnnealingAnnealing
 PCR does not copy the all of the DNA in the sample.PCR does not copy the all of the DNA in the sample.
 It copies only a very specific sequence of genetic code, targetedIt copies only a very specific sequence of genetic code, targeted
by the PCR primers.by the PCR primers.
 For example, Chlamydia has a unique pattern of nucleotidesFor example, Chlamydia has a unique pattern of nucleotides
specific to the bacteria. The PCR will copy only the specificspecific to the bacteria. The PCR will copy only the specific
DNA sequences that are present in Chlamydia and absent fromDNA sequences that are present in Chlamydia and absent from
other bacterial speciesother bacterial species
 To do this, PCR uses primers, man-made oligonucleotidesTo do this, PCR uses primers, man-made oligonucleotides
(short pieces of synthetic DNA) that bind, or anneal, only to(short pieces of synthetic DNA) that bind, or anneal, only to
sequences on either side of the target DNA region.sequences on either side of the target DNA region.
 Temperature of reaction mixture is cooled to 45-60 CTemperature of reaction mixture is cooled to 45-60 C
 This allows the primers to bind (anneal) to their complementaryThis allows the primers to bind (anneal) to their complementary
sequence in the template DNA.sequence in the template DNA.
 The two strands are ready to be copied.The two strands are ready to be copied.

8. ExtensionExtension
 In the third phase of the reaction, called extension, requires DNAIn the third phase of the reaction, called extension, requires DNA
synthesis by DNA polymerase.synthesis by DNA polymerase.
 To withstand the repeated exposure to high temperatures, aTo withstand the repeated exposure to high temperatures, a
thermostable DNA polymerase is used for PCR.thermostable DNA polymerase is used for PCR.
 A variety of thermostable DNA polymerases (isolated fromA variety of thermostable DNA polymerases (isolated from
different thermophilic Bacteria or Archae) are available, but thedifferent thermophilic Bacteria or Archae) are available, but the
first thermostable DNA polymerase used for PCR, called Taqfirst thermostable DNA polymerase used for PCR, called Taq
polymerase, was isolated from the bacterium Thermus aquaticus.polymerase, was isolated from the bacterium Thermus aquaticus.
 The optimal temperature for Taq polymerase is about 75-80oC,The optimal temperature for Taq polymerase is about 75-80oC,
but it is partially active even at typical annealing temperatures sobut it is partially active even at typical annealing temperatures so
primer extension begins during the annealing step.primer extension begins during the annealing step.
 The rate of primer extension by Taq polymerase is about 50-100The rate of primer extension by Taq polymerase is about 50-100
nucleotides/secnucleotides/sec

9. PCRPCR

10. Factors for optimal PCRFactors for optimal PCR
 >PCR Primers:>PCR Primers:
Correctly designed pair of primers is requiredCorrectly designed pair of primers is required
>DNA polymerase:>DNA polymerase:
Thermus aquaticus 170 FThermus aquaticus 170 F
Taq polymerase is heat resistantTaq polymerase is heat resistant
It lacks proof reading exonuclease activityIt lacks proof reading exonuclease activity
Other polymerases can be usedOther polymerases can be used
Tma DNA polymerase from Thermotoga maritamaTma DNA polymerase from Thermotoga maritama
Pfu DNa polymerase from Pyrococcus FuriosusPfu DNa polymerase from Pyrococcus Furiosus

11. Melting temp.Melting temp.
 Temp. at which 2 strands of duplex dissociateTemp. at which 2 strands of duplex dissociate
 It can be determined expermientallyIt can be determined expermientally
 Tm = (4(G+C) + (2(A+T)Tm = (4(G+C) + (2(A+T)

12. Types of polymerase chain
reaction

13.  Allele-specific PCR is a variation of the polymerase
chain reaction which is used as a diagnostic or cloning
technique, to identify or utilize single-nucleotide
polymorphisms (SNPs) (single base differences in
DNA)
 Allele-specific PCR is a modification of the general
PCR
Allele-specific polymerase chain
reaction.

14. Hot start PCR.
 The specificity and DNA yield of PCRs are
often improved by the “hot start” technique and
analogous method.
 It reduces non-specific priming,the formation of
primer dimers and increases product yield during
the initial set up stages of the PCR by
inactivating the taq polymerase at lower
temperature.

15.  It is one of the most commonly used polymerase chain
reaction used in molecular biology. It is a sensitive
method for the detection of mRNA expression levels.
 It involves two steps: RNA is first reverse transcribed
into cDNA using a reverse transcriptase and then the
resulting cDNA is used as templates for subsequent
PCR amplification using primers specific for one or
more genes.
Reverse transcription polymerase
chain reaction.

16. Quantitative polymerase chain
reaction.
Quantitative PCR is used widely to detect and
quantify specific DNA sequences in scientific
fields that range from fundamental biology to
biotechnology and forensic sciences. It
quantitatively measures starting amounts of
DNA, DNA, or RNA.

17. Multiplex-polymerase chain reaction.
 It uses multiple primer sets within a single PCR mixture to
produce amplicons of varying sizes that are specific to different
DNA sequences
 Annealing temperatures for each of the primer sets must be
optimized to work correctly within a single reaction, and
amplicon sizes. That is, their base pair length should be different
enough to form distinct bands when visualized by gel
electrophoresis
 Multiplex PCR was very useful in known mitochondrial DNA
mutations in Chinese patients with Leber’s hereditary optic
neuropathy (LHON). This disease is a maternally transmitted
disease
 An amplicon is piece of DNA or RNA that IS A source of
natural or artifical amplication or replication events.

18. Inverse polymerase chain reaction.
 In this method amplification of DNA of
unknown sequence is carried out from known
sequence
 It is especially used in identifying flanking
sequence of various genomic inserts.
 Genomic inserts are DNA’s exclusive of
duplications that occur between preexisting
genomic sequences.

19. Miniprimer polymerase chain
reaction.
 It uses “miniprimers” or “smalligos” which are
about 9 to 10 nucleotide s for detecting
sequences beyond those detected by standard
methods using longer primers and Taq
polymerase.
 By use of several combinations of miniprimers
PCRs 16S rRNA genes from Escherichia coli or
Halobacterium salinarum genomic DNA were
amplified.

20. GELGEL
ELECTROPHORESISELECTROPHORESIS

21. INTRODUCTIONINTRODUCTION
 ElectrophoresisElectrophoresis
 Electro refers to electron flow or current.Electro refers to electron flow or current.
 Phoresis refers to movement.Phoresis refers to movement.
 Thus Electrophoresis is movement under electricThus Electrophoresis is movement under electric
 current.current.
 This technique therefore can separate moleculesThis technique therefore can separate molecules
 which can move in an electric field i.e chargedwhich can move in an electric field i.e charged
 moleculesmolecules

22.
INTRODUCTIONINTRODUCTION
..
Gel Electrophrosis:-Gel Electrophrosis:-
 Gel electrophoresis is a laboratory method usedGel electrophoresis is a laboratory method used
to separate mixtures of DNA, RNA, or proteinsto separate mixtures of DNA, RNA, or proteins
according to molecular size.according to molecular size.

23. INTRODUCTIONINTRODUCTION
 In gel electrophoresis, the molecules to be separated areIn gel electrophoresis, the molecules to be separated are
pushed by an electrical field through a gel that containspushed by an electrical field through a gel that contains
small pores.small pores.
 Electrophoresis is a separations technique that is basedElectrophoresis is a separations technique that is based
on the the mobility of ions in an electric field. Positivelyon the the mobility of ions in an electric field. Positively
charged ions migrate towards a negative electrode andcharged ions migrate towards a negative electrode and
negatively-charged ions migrate toward a positivenegatively-charged ions migrate toward a positive
electrode.electrode.

24. BASIC PRINCIPALBASIC PRINCIPAL
 Its principle is that the charged particles of aIts principle is that the charged particles of a
sample migrate in an applied electrical field. Ifsample migrate in an applied electrical field. If
conducted in solution, samples are separatedconducted in solution, samples are separated
according to their surface net charge density.according to their surface net charge density.

25.
HISTORYHISTORY
 1930s - first reports of the use of sucrose for gel electrophoresis1930s - first reports of the use of sucrose for gel electrophoresis
 1955 - introduction of starch gels, mediocre separation1955 - introduction of starch gels, mediocre separation
 1959 - introduction of acrylamide gels (Raymond and Weintraub); accurate control of1959 - introduction of acrylamide gels (Raymond and Weintraub); accurate control of
parametersparameters
 such as pore size and stabilitysuch as pore size and stability
 1964 - disc gel electrophoresis (Ornstein and Davis)1964 - disc gel electrophoresis (Ornstein and Davis)
 1969 - introduction of denaturing agents especially SDS separation of protein subunit1969 - introduction of denaturing agents especially SDS separation of protein subunit
(Weber and osborn).(Weber and osborn).

26. Gel Electophrosis DNAGel Electophrosis DNA
 Gel electrophoresis detects the presence of DNA in a sampleGel electrophoresis detects the presence of DNA in a sample
 Gel electrophoresis detects the number of nucleotides in aGel electrophoresis detects the number of nucleotides in a
fragment of DNAfragment of DNA
 e.g., the number of nucleotides in a DNA region which wase.g., the number of nucleotides in a DNA region which was
amplified by PCRamplified by PCR
 Is a rough estimate, is not exact, need more sophisticatedIs a rough estimate, is not exact, need more sophisticated
sequencing techniques to get an exact number of nucleotidessequencing techniques to get an exact number of nucleotides
 Can be used to tentatively identify a gene because we know theCan be used to tentatively identify a gene because we know the
number of nucleotides in many genes.number of nucleotides in many genes.

27. Visualizing the DNA (QuikVIEW stain)
 250
 1,500
 1,000
 500
 750
 2,000 bp
DNA ladder

PCR
Product
wells
+ - - - - + + - - + - +
March 12, 2006
Samples # 1, 6, 7, 10 & 12 were positive for Wolbachia DNA

28. PROCEDUREPROCEDURE
 DNA is cut into smaller fragments.DNA is cut into smaller fragments.
 Loading dye is used to indicate the fragments ofLoading dye is used to indicate the fragments of
DNA are behind the dyeDNA are behind the dye
 The negative DNA molecule is attracted to theThe negative DNA molecule is attracted to the
positive electrode.positive electrode.
 The smallest fragments move the greatestThe smallest fragments move the greatest
distancedistance

29. PROCEDUREPROCEDURE
 Remove comb and observe wells.Remove comb and observe wells.
 Place carbon paper in each end of the tray.Place carbon paper in each end of the tray.
 Cover with buffer, making sure the allow buffer toCover with buffer, making sure the allow buffer to
overflow into each end of the tray.overflow into each end of the tray.
 Load gels.Load gels.
 Connect the electrodes.Connect the electrodes.
 Turn on power supply.Turn on power supply.
 Allow gels to run – make sure you see bubbles comingAllow gels to run – make sure you see bubbles coming
from the electrodesfrom the electrodes

30. PROCEDUREPROCEDURE
 It will take about 30 minutes for the gel to run.It will take about 30 minutes for the gel to run.
 Turn off power supply and remove electrodes.Turn off power supply and remove electrodes.
 Pour off buffer into the designated container.Pour off buffer into the designated container.
 Carefully remove gel from gel box and place inCarefully remove gel from gel box and place in
glad container and cover with stain.glad container and cover with stain.
 Store in appropriate locationStore in appropriate location

31. How Gel Electrophoresis DNA Works?How Gel Electrophoresis DNA Works?
 A sample which contains fragments of DNA is forced by an electrical currentA sample which contains fragments of DNA is forced by an electrical current
through a firm gel which is really a sieve with small holes of a fixed size.through a firm gel which is really a sieve with small holes of a fixed size.
 Phosphate group in DNA is negatively charged so it is moved towards aPhosphate group in DNA is negatively charged so it is moved towards a
positive electrode by the current.positive electrode by the current.
 Longer fragments have more nucleotides.Longer fragments have more nucleotides.
 So have a larger molecular weight.So have a larger molecular weight.
 So are bigger in size.So are bigger in size.
 So aren’t able to pass through the small holes in the gel and get hung up atSo aren’t able to pass through the small holes in the gel and get hung up at
the beginning of the gel.the beginning of the gel.
 A sample which contains fragments of DNA is forced by an electrical currentA sample which contains fragments of DNA is forced by an electrical current
through a firm gel which is really a sieve with small holes of a fixed size.through a firm gel which is really a sieve with small holes of a fixed size.
 Phosphate group in DNA is negatively charged so it is moved towards aPhosphate group in DNA is negatively charged so it is moved towards a
positive electrode by the current.positive electrode by the current.
 Longer fragments have more nucleotides.Longer fragments have more nucleotides.
 So have a larger molecular weight.So have a larger molecular weight.
 So are bigger in size.So are bigger in size.
 So aren’t able to pass through the small holes in the gel and get hung up atSo aren’t able to pass through the small holes in the gel and get hung up at
the beginning of the gel.the beginning of the gel.

32. Conti……Conti……
 Shorter fragments are able to pass through and moveShorter fragments are able to pass through and move
farther along the gel.farther along the gel.
 Fragments of intermediate length travel to about theFragments of intermediate length travel to about the
middle of the gel.middle of the gel.
 DNA fragments are then visualized in the gel with aDNA fragments are then visualized in the gel with a
special dye.special dye.
 The number of nucleotides are then estimated byThe number of nucleotides are then estimated by
comparing it to a known sample of DNA fragmentscomparing it to a known sample of DNA fragments
which is run through the gel at the same time.which is run through the gel at the same time.

33. REAGENTS NEEDEDREAGENTS NEEDED
 Sample of DNA fragmentsSample of DNA fragments
 Known sample of DNA fragmentsKnown sample of DNA fragments
 DNA ladderDNA ladder
 GelGel
 AgaroseAgarose
 Dye to visualize the movement of the sample as it is travelingDye to visualize the movement of the sample as it is traveling
through the gelthrough the gel
 Loading dye – Blue juiceLoading dye – Blue juice
 BufferBuffer

34. Equipment NeededEquipment Needed
 Box to hold the gelBox to hold the gel
 Comb to create small wells in the agarose gel toComb to create small wells in the agarose gel to
put the DNA sample into at the beginning ofput the DNA sample into at the beginning of
the gelthe gel
 Positive and negative electrodes to create thePositive and negative electrodes to create the
electrical currentelectrical current
 Power supplyPower supply
 Gel photo imaging systemGel photo imaging system

36. Types of GelTypes of Gel
ElectrophoresisElectrophoresis

37. Some media for ElectrophoresisSome media for Electrophoresis
MediumMedium ConditionsConditions Principal UsesPrincipal Uses
PaperPaper Filter paper moistenedFilter paper moistened
With Buffer, placedWith Buffer, placed
Between electrodsBetween electrods
Small moleculesSmall molecules
Amino acid, nucleotidesAmino acid, nucleotides
Polyacrylamide gelPolyacrylamide gel Cast in tubes or slabs;Cast in tubes or slabs; Proteins and nucleic acidsProteins and nucleic acids
Agarose gelAgarose gel As polyacrylamide,As polyacrylamide, Very large proteins,Very large proteins,
Nucleic acid andNucleic acid and
Nucleoprotiens etcNucleoprotiens etc

38.  There are two types of gel ElectrophoresisThere are two types of gel Electrophoresis
 →→ One dimensionOne dimension
 →→ Two dimensionsTwo dimensions

39. One dimensionOne dimension
 1. SDS-PAGE,1. SDS-PAGE,
 2. Native –PAGE2. Native –PAGE
 3. IE3. IE
 Several forms of PAGE exist and can provide different types of informationSeveral forms of PAGE exist and can provide different types of information
aboutabout
 the protein(s).the protein(s).
 SDS-PAGE, the most widely used electrophoresis technique, separates proteinsSDS-PAGE, the most widely used electrophoresis technique, separates proteins
 primarily by mass.primarily by mass.
 Non denaturing PAGE, also called native PAGE, separates proteins according toNon denaturing PAGE, also called native PAGE, separates proteins according to
 their mass:charge ratio.their mass:charge ratio.
 Two-dimensional PAGE (2D-PAGE) separates proteins by isoelectric point in theTwo-dimensional PAGE (2D-PAGE) separates proteins by isoelectric point in the
 first dimension and by mass in the second dimension.first dimension and by mass in the second dimension.

40. Agarose gel electrophoresisAgarose gel electrophoresis
Agarose gel electrophoresisAgarose gel electrophoresis is a method tois a method to
separate DNA, or RNA molecules by size. Thisseparate DNA, or RNA molecules by size. This
is achieved by moving negatively charged nucleicis achieved by moving negatively charged nucleic
acid molecules through an agarose matrix withacid molecules through an agarose matrix with
an electric field (electrophoresis). Shorteran electric field (electrophoresis). Shorter
molecules move faster and migrate farther thanmolecules move faster and migrate farther than
longer ones .longer ones .
Analysis of PCR products, e.g. in molecularAnalysis of PCR products, e.g. in molecular
genetic diagnosis or genetic fingerprintinggenetic diagnosis or genetic fingerprinting

41.  Agarose is a highly purified uncharged polysaccharideAgarose is a highly purified uncharged polysaccharide
derived from agarderived from agar
 Agarose dissolves when added to boiling liquid. ItAgarose dissolves when added to boiling liquid. It
remains in a liquid state until the temperature isremains in a liquid state until the temperature is
lowered to about 40° C at which point it gelslowered to about 40° C at which point it gels
 The pore size may be predetermined by adjusting theThe pore size may be predetermined by adjusting the
concentration of agarose in the gelconcentration of agarose in the gel
 Agarose gels are fragile, however. They are actuallyAgarose gels are fragile, however. They are actually
hydrocolloids, and they are held together by thehydrocolloids, and they are held together by the
formation of weak hydrogen and hydrophobic bondsformation of weak hydrogen and hydrophobic bonds

42. Structure of the Repeating Unit ofStructure of the Repeating Unit of
Agarose, 3,6-anhydro-L-galactoseAgarose, 3,6-anhydro-L-galactose
Basic
disaccharide
repeating units of
agarose,
G: 1,3-β-d-
galactose
and
A: 1,4-α-l-3,6-
anhydrogalactose

43. Gel Structure of AgaroseGel Structure of Agarose

44. How fast will the DNA migrate?

45. Polyacrylamide GelsPolyacrylamide Gels
 Polyacrylamide gels are tougher thanPolyacrylamide gels are tougher than
agarose gelsagarose gels
 Acrylamide monomers polymerize into longAcrylamide monomers polymerize into long
chains that are covalently linked by achains that are covalently linked by a
crosslinkercrosslinker
 Polyacrylamide is chemically complex, as isPolyacrylamide is chemically complex, as is
the production and use of the gelthe production and use of the gel

46. How does an SDS-PAGE gelHow does an SDS-PAGE gel
work?work?
•Negatively charged
proteins move to
positive electrode
•Smaller proteins
move faster
• Proteins separate
by size
-
+
s-s
SDS, heat
proteins with
SDS

47. Muscle Contains Proteins ofMuscle Contains Proteins of
Many SizesMany Sizes
ProteinProtein kDakDa FunctionFunction
titintitin 30003000 center myosin in sarcomerecenter myosin in sarcomere
dystrophindystrophin 400400 anchoring to plasma membraneanchoring to plasma membrane
filaminfilamin 270270 cross-link filaments into gelcross-link filaments into gel
myosinmyosin heavy chainheavy chain 210210 slide filamentsslide filaments
spectrinspectrin 265265 attach filaments to plasmaattach filaments to plasma
membranemembrane
nebulinnebulin 107107 regulate actin assemblyregulate actin assembly
αα-actinin-actinin 100100 bundle filamentsbundle filaments
gelosingelosin 9090 fragment filamentsfragment filaments
fimbrinfimbrin 6868 bundle filamentsbundle filaments
actinactin 4242 form filamentsform filaments
tropomyosintropomyosin 3535 strengthen filamentsstrengthen filaments
myosinmyosin light chainlight chain 2727 slide filamentsslide filaments
troponin (T, I, C)troponin (T, I, C) 30, 19, 1730, 19, 17 mediate regulation of contractionmediate regulation of contraction
thymosinthymosin 55 sequester actin monomerssequester actin monomers

48. Isoelectric PointIsoelectric Point
 There is a pH at which there is no net chargeThere is a pH at which there is no net charge
on a protein; this is the isoelectric point.on a protein; this is the isoelectric point.
 Above its isoelectric point, a protein has a netAbove its isoelectric point, a protein has a net
negative charge and migrates toward thenegative charge and migrates toward the
anode in an electrical field.anode in an electrical field.
 Below its isoelectric point, the protein isBelow its isoelectric point, the protein is
positive and migrates toward the cathode.positive and migrates toward the cathode.

50. Isoelectric FocusingIsoelectric Focusing
 Isoelectric focusing is a method in which proteins areIsoelectric focusing is a method in which proteins are
separated in a pH gradient according to their isoelectricseparated in a pH gradient according to their isoelectric
pointspoints
 Focusing occurs in two stages; first, the pH gradient isFocusing occurs in two stages; first, the pH gradient is
formedformed
 In the second stage, the proteins begin their migrationsIn the second stage, the proteins begin their migrations
toward the anode if their net charge is negative, ortoward the anode if their net charge is negative, or
toward the cathode if their net charge is positivetoward the cathode if their net charge is positive
 When a protein reaches its isoelectric point (pI) in theWhen a protein reaches its isoelectric point (pI) in the
pH gradient, it carries a net charge of zero and will stoppH gradient, it carries a net charge of zero and will stop
migratingmigrating

51. Isoelectric FocusingIsoelectric Focusing

52. What is DNA Profiling?What is DNA Profiling?
A technique used by scientists to distinguishA technique used by scientists to distinguish
between individuals of the same species usingbetween individuals of the same species using
only samples of their DNAonly samples of their DNA

53. Stages of DNA ProfilingStages of DNA Profiling
 Step :Step :
The DNA is cut into fragments usingThe DNA is cut into fragments using restriction enzymesrestriction enzymes..
Each restriction enzyme cuts DNA at a specific base sequence.Each restriction enzyme cuts DNA at a specific base sequence.

54. Stages of DNA ProfilingStages of DNA Profiling
 A radioactive material isA radioactive material is
added which combinesadded which combines
with the DNA fragmentswith the DNA fragments
to produce a fluorescentto produce a fluorescent
image.image.
 A photographic copy ofA photographic copy of
the DNA bands isthe DNA bands is
obtained.obtained.

55. Stages of DNA ProfilingStages of DNA Profiling
Stage :Stage :
 The pattern of fragment distribution is thenThe pattern of fragment distribution is then
analysed.analysed.

56. Uses of DNA ProfilingUses of DNA Profiling
 DNA profiling isDNA profiling is
used to solveused to solve crimescrimes
andand medicalmedical
problemsproblems

57. ApplicationsApplications

58. Applications of gel electrophoresisApplications of gel electrophoresis
 Can be used for:Can be used for:
 Analytical purposesAnalytical purposes : often after amplification of: often after amplification of
DNA via PCRDNA via PCR
 preparative purposespreparative purposes : prior to use of other: prior to use of other
methods such as southern blotting ,cloningmethods such as southern blotting ,cloning
,PCR for further characterization.,PCR for further characterization.
 Gel electrophoresis can also be used forGel electrophoresis can also be used for
separation of nano particles.separation of nano particles.

59.  Check the quality and quantity of genomic DNACheck the quality and quantity of genomic DNA
after after DNA extractionDNA extraction
 Separate DNA fragments to clone a specificSeparate DNA fragments to clone a specific
DNA segment. DNA segment.

60. APPLICATIONS OF PCRAPPLICATIONS OF PCR
 PCR has applications in many fields of geneticPCR has applications in many fields of genetic
analysis:analysis:
 Medical applications:Medical applications:
 Genetic testing:for the identification of geneticGenetic testing:for the identification of genetic
diseases and carriers like thalassemia anddiseases and carriers like thalassemia and
prenatal testingprenatal testing
 tissue typing: organ transplantation.tissue typing: organ transplantation.
 Oncogenes:for the identification of geneOncogenes:for the identification of gene
mutations.mutations.

61.  Infectious diseases:for the diagnosis andInfectious diseases:for the diagnosis and
treatment of infectious diseases like HIVtreatment of infectious diseases like HIV
&TB &TB
 FORENSIC APPLICATIONS:FORENSIC APPLICATIONS:
 Genetic fingerprinting:Genetic fingerprinting:
 DNA fingerprinting for the identification ofDNA fingerprinting for the identification of
biological parentsbiological parents

62.  RESEARCH APPLICATIONSRESEARCH APPLICATIONS::
 PCR is used in many research procedure likePCR is used in many research procedure like
DNA cloning,genetic mapping,DNA sequencingDNA cloning,genetic mapping,DNA sequencing
by rapid production of short segmants of DNA.by rapid production of short segmants of DNA.