SlideShare ist ein Scribd-Unternehmen logo
1 von 17
Restriction Fragment
Length Polymorphism
(RFLP)
Group Members:
Ashfaq Ahmad (08)
Pirzad (91)
Basit Ali (38)
Muhammad Saleh
What is RFLP ?
 Restriction Fragment Length Polymorphism is a variation
in the length of a DNA fragment produced by a specific
restriction enzyme acting on a DNA from different
individuals that usually results from a genetic mutation.
 If two organisms differ in the distance between site of
cleavage of a particular restriction endonuclease, the
length of the fragments produced will be different when
the DNA is digested with a restriction enzyme.
 RFLP analysis is the detection of the change in the
length of the restriction fragments.
Continuous
 A restriction enzyme cuts the DNA molecule at every
occurrence of a particular sequence, called restriction
site.
 For Example, HindIII enzyme cuts at AAGCTT.
 If we apply a restriction enzyme on DNA, it is cut every
occurrence of the restriction site into a million
restriction fragments each a few thousands nucleotides
long.
Continuous
 Any mutation or polymorphism of a single nucleotide may
destroy (AAGCTT for HindIII) and change the length
of the fragment.
 The term polymorphism refers to the slight differences
between individuals, in base pair sequences of genes
or
 A polymorphism is a clinically harmless DNA variation
that does not affect the phenotype.
 So RFLP analysis is a technique which is used to detect
the change in the length of the restriction fragments.
RFLP Technology
 RFLP detection depends on the possibility of comparing
band profiles generated after restriction enzyme
digestion of target DNA. These differences in fragment
lengths can be seen after gel electrophoresis,
hybridization and visualization. The basic steps involved
are as follows:
1. Isolation of DNA
2. Restriction Digestion & Gel Electrophoresis
3. DNA transfer by Southern blotting
4. DNA hybridization
(1) Isolation of DNA
 Isolating DNA is the first step for many DNA-based
technologies. DNA is found either in nuclear
chromosomes or in organelles (mitochondria and
chloroplast).
 To extract DNA from its location, several laboratory
procedures are needed to break the cell wall and nuclear
membrane, and so appropriately separate the DNA from
other cell components.
 When doing so, care must be taken to ensure the
process does not damage the DNA molecule and that it
is recovered in the form of a long thread.
(2) Restriction Digestion & Gel
Electrophoresis
 Extracted DNA is digested with specific, carefully
chosen, restriction enzymes.
 Each restriction enzyme, under suitable conditions, will
recognize and cut DNA resulting in a restriction
fragments of different lengths.
 The thousands of restriction fragments produced are
commonly separated by Gel Electrophoresis on agarose
gels.
Continuous
 For visualization of DNA bands on the gel, it is stained
with ethidium bromide. But staining alone cannot detect
polymorphisms.
 Hybridization must therefore be used to detect specific
fragments.
(3) DNA transfer by Southern
blotting
 DNA transfer is called “Southern blotting”, after E.M.
Southern (1975), who invented the technique.
 In this method, the gel is first denatured in a basic
solution of NaOH and placed in a tray. A porous Nylon or
nitrocellulose membrane is laid over the gel.
 All the DNA restriction fragments in the gel transfer as
single strands by capillary action to the membrane. All
fragments retain the same pattern on the membrane as
on the gel.
(4) DNA Hybridization
 The membrane with the target DNA is exposed to the
DNA probe (radioactively labelled). On the basis of
availability and complementarity, hybridization will
occur.
 The DNA probe is a singe-stranded molecule,
conveniently labelled, using any standard method (e.g. a
radioisotope), and hybridize with the target DNA, which
is stuck to the membrane.
Continuous
 Thus the DNA probe binds with the sequences that are
complementary to it among the thousands or millions of
undetected fragments that migrate through the gel.
 Desired fragments maybe detected after simultaneous
exposure of the hybridized probe to X-rays, which on
exposure appear as black spots on the X-ray film.
Applications of RFLP
 The important applications of RFLP are:
1. RFLP can be used paternity cases or criminal cases to
determine the source of a DNA sample (i.e. it has
forensic applications).
2. RFLP can be used to detect mutations.
Advantages of RFLP
 Some of the advantages of RFLP are as follow:
1. It is a simple process.
2. It is an accurate process.
3. No sequence information required.
Disadvantages of RFLP
 Some of the disadvantages of RFLP are as follow:
1. It is an expensive process.
2. It is slow and time consuming process.
3. Labour intensive.
4. Requires very large amount of DNA.
5. It is difficult to automate.
Rflp presentation

Weitere ähnliche Inhalte

Was ist angesagt?

Chromosome walking
Chromosome walkingChromosome walking
Chromosome walking
Aleena Khan
 
Complementary DNA (cDNA) Libraries
Complementary DNA 	(cDNA) LibrariesComplementary DNA 	(cDNA) Libraries
Complementary DNA (cDNA) Libraries
Ramesh Pothuraju
 

Was ist angesagt? (20)

Dna sequencing
Dna sequencingDna sequencing
Dna sequencing
 
Rapd ppt
Rapd pptRapd ppt
Rapd ppt
 
AFLP, RFLP & RAPD
AFLP, RFLP & RAPDAFLP, RFLP & RAPD
AFLP, RFLP & RAPD
 
Sanger sequencing
Sanger sequencing Sanger sequencing
Sanger sequencing
 
DNA Sequencing
DNA SequencingDNA Sequencing
DNA Sequencing
 
Pcr and its applications
Pcr and its applicationsPcr and its applications
Pcr and its applications
 
dna sequencing methods
 dna sequencing methods dna sequencing methods
dna sequencing methods
 
Sanger sequencing (DNA sequencing by ENZYMATIC METHOD)
Sanger sequencing (DNA sequencing by ENZYMATIC METHOD)Sanger sequencing (DNA sequencing by ENZYMATIC METHOD)
Sanger sequencing (DNA sequencing by ENZYMATIC METHOD)
 
Dna sequencing
Dna sequencingDna sequencing
Dna sequencing
 
Chromosome walking
Chromosome walkingChromosome walking
Chromosome walking
 
Restriction Mapping
Restriction MappingRestriction Mapping
Restriction Mapping
 
Gene library
Gene libraryGene library
Gene library
 
Random Amplified polymorphic DNA. RAPD
Random Amplified polymorphic DNA. RAPDRandom Amplified polymorphic DNA. RAPD
Random Amplified polymorphic DNA. RAPD
 
Maxam-Gilbert method of DNA sequencing
Maxam-Gilbert method of DNA sequencingMaxam-Gilbert method of DNA sequencing
Maxam-Gilbert method of DNA sequencing
 
Different pcr techniques and their application
Different pcr techniques and their applicationDifferent pcr techniques and their application
Different pcr techniques and their application
 
Complementary DNA (cDNA) Libraries
Complementary DNA 	(cDNA) LibrariesComplementary DNA 	(cDNA) Libraries
Complementary DNA (cDNA) Libraries
 
DNA footprinting
DNA footprintingDNA footprinting
DNA footprinting
 
Gene isolation methods
Gene isolation methodsGene isolation methods
Gene isolation methods
 
Genome mapping
Genome mapping Genome mapping
Genome mapping
 
Pyrosequencing
PyrosequencingPyrosequencing
Pyrosequencing
 

Ähnlich wie Rflp presentation

Human genome project
Human genome projectHuman genome project
Human genome project
15cookho
 
Lecture 5 dna finger, foot printing rflp
Lecture 5 dna finger, foot printing rflpLecture 5 dna finger, foot printing rflp
Lecture 5 dna finger, foot printing rflp
Dr Vishnu Kumar
 
Biotech 2011-06-electrophoresis-blots
Biotech 2011-06-electrophoresis-blotsBiotech 2011-06-electrophoresis-blots
Biotech 2011-06-electrophoresis-blots
Nikolay Vyahhi
 
Biotech 2011-06-electrophoresis-blots
Biotech 2011-06-electrophoresis-blotsBiotech 2011-06-electrophoresis-blots
Biotech 2011-06-electrophoresis-blots
Nikolay Vyahhi
 
DNA FINGERPRINTING
DNA FINGERPRINTINGDNA FINGERPRINTING
DNA FINGERPRINTING
Parth Shah
 
Copy Of Dna Sequencing
Copy Of Dna SequencingCopy Of Dna Sequencing
Copy Of Dna Sequencing
Zahoor Ahmed
 
Dna chips, RFLPs & dna fingerprint
Dna chips, RFLPs & dna fingerprintDna chips, RFLPs & dna fingerprint
Dna chips, RFLPs & dna fingerprint
Tapeshwar Yadav
 
PPTChapter 6 Molecular Basis of Inheritance G.pptx
PPTChapter 6 Molecular Basis of Inheritance G.pptxPPTChapter 6 Molecular Basis of Inheritance G.pptx
PPTChapter 6 Molecular Basis of Inheritance G.pptx
MaryDiana27
 
Getitics Slides #1 - Modified
Getitics Slides #1 - Modified Getitics Slides #1 - Modified
Getitics Slides #1 - Modified
Ahmad B. Younes
 

Ähnlich wie Rflp presentation (20)

Rflp technology
Rflp technologyRflp technology
Rflp technology
 
Human genome project
Human genome projectHuman genome project
Human genome project
 
Molecular Techniques in Microbiology.pptx
Molecular Techniques in Microbiology.pptxMolecular Techniques in Microbiology.pptx
Molecular Techniques in Microbiology.pptx
 
Lecture 5 dna finger, foot printing rflp
Lecture 5 dna finger, foot printing rflpLecture 5 dna finger, foot printing rflp
Lecture 5 dna finger, foot printing rflp
 
Biotech 2011-06-electrophoresis-blots
Biotech 2011-06-electrophoresis-blotsBiotech 2011-06-electrophoresis-blots
Biotech 2011-06-electrophoresis-blots
 
Biotech 2011-06-electrophoresis-blots
Biotech 2011-06-electrophoresis-blotsBiotech 2011-06-electrophoresis-blots
Biotech 2011-06-electrophoresis-blots
 
RFLP
RFLP RFLP
RFLP
 
DNA FINGERPRINTING
DNA FINGERPRINTINGDNA FINGERPRINTING
DNA FINGERPRINTING
 
Copy Of Dna Sequencing
Copy Of Dna SequencingCopy Of Dna Sequencing
Copy Of Dna Sequencing
 
Dna chips, RFLPs & dna fingerprint
Dna chips, RFLPs & dna fingerprintDna chips, RFLPs & dna fingerprint
Dna chips, RFLPs & dna fingerprint
 
Biotechnology: Principles and Processes Class XII Chapter 11.pptx
Biotechnology: Principles and Processes Class XII Chapter 11.pptxBiotechnology: Principles and Processes Class XII Chapter 11.pptx
Biotechnology: Principles and Processes Class XII Chapter 11.pptx
 
PPTChapter 6 Molecular Basis of Inheritance G.pptx
PPTChapter 6 Molecular Basis of Inheritance G.pptxPPTChapter 6 Molecular Basis of Inheritance G.pptx
PPTChapter 6 Molecular Basis of Inheritance G.pptx
 
PPTChapter 6 Molecular Basis of Inheritance G (1).pptx
PPTChapter 6 Molecular Basis of Inheritance G (1).pptxPPTChapter 6 Molecular Basis of Inheritance G (1).pptx
PPTChapter 6 Molecular Basis of Inheritance G (1).pptx
 
Restriction mapping
Restriction mappingRestriction mapping
Restriction mapping
 
Restriction Digestion and its Applications
Restriction Digestion and its ApplicationsRestriction Digestion and its Applications
Restriction Digestion and its Applications
 
RFLP.pdf
RFLP.pdfRFLP.pdf
RFLP.pdf
 
Restriction enzymes
Restriction enzymesRestriction enzymes
Restriction enzymes
 
Rflp
RflpRflp
Rflp
 
Nucleic acid hybridization
Nucleic acid hybridizationNucleic acid hybridization
Nucleic acid hybridization
 
Getitics Slides #1 - Modified
Getitics Slides #1 - Modified Getitics Slides #1 - Modified
Getitics Slides #1 - Modified
 

Mehr von Ashfaq Ahmad

Mehr von Ashfaq Ahmad (20)

10000 plus English Vocabulary
10000 plus English Vocabulary10000 plus English Vocabulary
10000 plus English Vocabulary
 
Personality and psychographics
Personality and psychographicsPersonality and psychographics
Personality and psychographics
 
Affinity chromatography
Affinity chromatographyAffinity chromatography
Affinity chromatography
 
Basics of spectroscopy
Basics of spectroscopyBasics of spectroscopy
Basics of spectroscopy
 
Spectroscopy basics
Spectroscopy basicsSpectroscopy basics
Spectroscopy basics
 
High performance liquid chromatography
High performance liquid chromatographyHigh performance liquid chromatography
High performance liquid chromatography
 
Affinity chromatography and gel filteration
Affinity chromatography and gel filterationAffinity chromatography and gel filteration
Affinity chromatography and gel filteration
 
Lecture 11 and 12 microbial_sem_6 (1)
Lecture 11 and 12 microbial_sem_6 (1)Lecture 11 and 12 microbial_sem_6 (1)
Lecture 11 and 12 microbial_sem_6 (1)
 
Lecture 9 and 10 microbial_sem_6
Lecture 9 and 10 microbial_sem_6Lecture 9 and 10 microbial_sem_6
Lecture 9 and 10 microbial_sem_6
 
Lecture 7 and 8 microbial_sem_6_20180307
Lecture 7 and 8 microbial_sem_6_20180307Lecture 7 and 8 microbial_sem_6_20180307
Lecture 7 and 8 microbial_sem_6_20180307
 
Lecture 5 and 6 microbial_sem_6_20180307
Lecture 5 and 6 microbial_sem_6_20180307Lecture 5 and 6 microbial_sem_6_20180307
Lecture 5 and 6 microbial_sem_6_20180307
 
Chromatography basics
Chromatography basicsChromatography basics
Chromatography basics
 
Research methodology notes
Research methodology notesResearch methodology notes
Research methodology notes
 
Lecture 2 microbial_sem_6_20180220
Lecture 2 microbial_sem_6_20180220Lecture 2 microbial_sem_6_20180220
Lecture 2 microbial_sem_6_20180220
 
Lecture 1 microbial_sem_6_20170213
Lecture 1 microbial_sem_6_20170213Lecture 1 microbial_sem_6_20170213
Lecture 1 microbial_sem_6_20170213
 
Western blotting
Western blottingWestern blotting
Western blotting
 
Structural genomics
Structural genomicsStructural genomics
Structural genomics
 
Structural genomics
Structural genomicsStructural genomics
Structural genomics
 
Snp and its role in diseases
Snp and its role in diseasesSnp and its role in diseases
Snp and its role in diseases
 
Immunostaining
ImmunostainingImmunostaining
Immunostaining
 

Kürzlich hochgeladen

The basics of sentences session 3pptx.pptx
The basics of sentences session 3pptx.pptxThe basics of sentences session 3pptx.pptx
The basics of sentences session 3pptx.pptx
heathfieldcps1
 
Salient Features of India constitution especially power and functions
Salient Features of India constitution especially power and functionsSalient Features of India constitution especially power and functions
Salient Features of India constitution especially power and functions
KarakKing
 

Kürzlich hochgeladen (20)

This PowerPoint helps students to consider the concept of infinity.
This PowerPoint helps students to consider the concept of infinity.This PowerPoint helps students to consider the concept of infinity.
This PowerPoint helps students to consider the concept of infinity.
 
ICT role in 21st century education and it's challenges.
ICT role in 21st century education and it's challenges.ICT role in 21st century education and it's challenges.
ICT role in 21st century education and it's challenges.
 
The basics of sentences session 3pptx.pptx
The basics of sentences session 3pptx.pptxThe basics of sentences session 3pptx.pptx
The basics of sentences session 3pptx.pptx
 
Basic Civil Engineering first year Notes- Chapter 4 Building.pptx
Basic Civil Engineering first year Notes- Chapter 4 Building.pptxBasic Civil Engineering first year Notes- Chapter 4 Building.pptx
Basic Civil Engineering first year Notes- Chapter 4 Building.pptx
 
Single or Multiple melodic lines structure
Single or Multiple melodic lines structureSingle or Multiple melodic lines structure
Single or Multiple melodic lines structure
 
Understanding Accommodations and Modifications
Understanding  Accommodations and ModificationsUnderstanding  Accommodations and Modifications
Understanding Accommodations and Modifications
 
Micro-Scholarship, What it is, How can it help me.pdf
Micro-Scholarship, What it is, How can it help me.pdfMicro-Scholarship, What it is, How can it help me.pdf
Micro-Scholarship, What it is, How can it help me.pdf
 
General Principles of Intellectual Property: Concepts of Intellectual Proper...
General Principles of Intellectual Property: Concepts of Intellectual  Proper...General Principles of Intellectual Property: Concepts of Intellectual  Proper...
General Principles of Intellectual Property: Concepts of Intellectual Proper...
 
80 ĐỀ THI THỬ TUYỂN SINH TIẾNG ANH VÀO 10 SỞ GD – ĐT THÀNH PHỐ HỒ CHÍ MINH NĂ...
80 ĐỀ THI THỬ TUYỂN SINH TIẾNG ANH VÀO 10 SỞ GD – ĐT THÀNH PHỐ HỒ CHÍ MINH NĂ...80 ĐỀ THI THỬ TUYỂN SINH TIẾNG ANH VÀO 10 SỞ GD – ĐT THÀNH PHỐ HỒ CHÍ MINH NĂ...
80 ĐỀ THI THỬ TUYỂN SINH TIẾNG ANH VÀO 10 SỞ GD – ĐT THÀNH PHỐ HỒ CHÍ MINH NĂ...
 
HMCS Max Bernays Pre-Deployment Brief (May 2024).pptx
HMCS Max Bernays Pre-Deployment Brief (May 2024).pptxHMCS Max Bernays Pre-Deployment Brief (May 2024).pptx
HMCS Max Bernays Pre-Deployment Brief (May 2024).pptx
 
Wellbeing inclusion and digital dystopias.pptx
Wellbeing inclusion and digital dystopias.pptxWellbeing inclusion and digital dystopias.pptx
Wellbeing inclusion and digital dystopias.pptx
 
Holdier Curriculum Vitae (April 2024).pdf
Holdier Curriculum Vitae (April 2024).pdfHoldier Curriculum Vitae (April 2024).pdf
Holdier Curriculum Vitae (April 2024).pdf
 
How to Create and Manage Wizard in Odoo 17
How to Create and Manage Wizard in Odoo 17How to Create and Manage Wizard in Odoo 17
How to Create and Manage Wizard in Odoo 17
 
Salient Features of India constitution especially power and functions
Salient Features of India constitution especially power and functionsSalient Features of India constitution especially power and functions
Salient Features of India constitution especially power and functions
 
Food safety_Challenges food safety laboratories_.pdf
Food safety_Challenges food safety laboratories_.pdfFood safety_Challenges food safety laboratories_.pdf
Food safety_Challenges food safety laboratories_.pdf
 
Towards a code of practice for AI in AT.pptx
Towards a code of practice for AI in AT.pptxTowards a code of practice for AI in AT.pptx
Towards a code of practice for AI in AT.pptx
 
Beyond_Borders_Understanding_Anime_and_Manga_Fandom_A_Comprehensive_Audience_...
Beyond_Borders_Understanding_Anime_and_Manga_Fandom_A_Comprehensive_Audience_...Beyond_Borders_Understanding_Anime_and_Manga_Fandom_A_Comprehensive_Audience_...
Beyond_Borders_Understanding_Anime_and_Manga_Fandom_A_Comprehensive_Audience_...
 
SOC 101 Demonstration of Learning Presentation
SOC 101 Demonstration of Learning PresentationSOC 101 Demonstration of Learning Presentation
SOC 101 Demonstration of Learning Presentation
 
Key note speaker Neum_Admir Softic_ENG.pdf
Key note speaker Neum_Admir Softic_ENG.pdfKey note speaker Neum_Admir Softic_ENG.pdf
Key note speaker Neum_Admir Softic_ENG.pdf
 
Unit 3 Emotional Intelligence and Spiritual Intelligence.pdf
Unit 3 Emotional Intelligence and Spiritual Intelligence.pdfUnit 3 Emotional Intelligence and Spiritual Intelligence.pdf
Unit 3 Emotional Intelligence and Spiritual Intelligence.pdf
 

Rflp presentation

  • 1.
  • 2. Restriction Fragment Length Polymorphism (RFLP) Group Members: Ashfaq Ahmad (08) Pirzad (91) Basit Ali (38) Muhammad Saleh
  • 3. What is RFLP ?  Restriction Fragment Length Polymorphism is a variation in the length of a DNA fragment produced by a specific restriction enzyme acting on a DNA from different individuals that usually results from a genetic mutation.  If two organisms differ in the distance between site of cleavage of a particular restriction endonuclease, the length of the fragments produced will be different when the DNA is digested with a restriction enzyme.  RFLP analysis is the detection of the change in the length of the restriction fragments.
  • 4. Continuous  A restriction enzyme cuts the DNA molecule at every occurrence of a particular sequence, called restriction site.  For Example, HindIII enzyme cuts at AAGCTT.  If we apply a restriction enzyme on DNA, it is cut every occurrence of the restriction site into a million restriction fragments each a few thousands nucleotides long.
  • 5. Continuous  Any mutation or polymorphism of a single nucleotide may destroy (AAGCTT for HindIII) and change the length of the fragment.  The term polymorphism refers to the slight differences between individuals, in base pair sequences of genes or  A polymorphism is a clinically harmless DNA variation that does not affect the phenotype.  So RFLP analysis is a technique which is used to detect the change in the length of the restriction fragments.
  • 6. RFLP Technology  RFLP detection depends on the possibility of comparing band profiles generated after restriction enzyme digestion of target DNA. These differences in fragment lengths can be seen after gel electrophoresis, hybridization and visualization. The basic steps involved are as follows: 1. Isolation of DNA 2. Restriction Digestion & Gel Electrophoresis 3. DNA transfer by Southern blotting 4. DNA hybridization
  • 7. (1) Isolation of DNA  Isolating DNA is the first step for many DNA-based technologies. DNA is found either in nuclear chromosomes or in organelles (mitochondria and chloroplast).  To extract DNA from its location, several laboratory procedures are needed to break the cell wall and nuclear membrane, and so appropriately separate the DNA from other cell components.  When doing so, care must be taken to ensure the process does not damage the DNA molecule and that it is recovered in the form of a long thread.
  • 8. (2) Restriction Digestion & Gel Electrophoresis  Extracted DNA is digested with specific, carefully chosen, restriction enzymes.  Each restriction enzyme, under suitable conditions, will recognize and cut DNA resulting in a restriction fragments of different lengths.  The thousands of restriction fragments produced are commonly separated by Gel Electrophoresis on agarose gels.
  • 9. Continuous  For visualization of DNA bands on the gel, it is stained with ethidium bromide. But staining alone cannot detect polymorphisms.  Hybridization must therefore be used to detect specific fragments.
  • 10. (3) DNA transfer by Southern blotting  DNA transfer is called “Southern blotting”, after E.M. Southern (1975), who invented the technique.  In this method, the gel is first denatured in a basic solution of NaOH and placed in a tray. A porous Nylon or nitrocellulose membrane is laid over the gel.  All the DNA restriction fragments in the gel transfer as single strands by capillary action to the membrane. All fragments retain the same pattern on the membrane as on the gel.
  • 11. (4) DNA Hybridization  The membrane with the target DNA is exposed to the DNA probe (radioactively labelled). On the basis of availability and complementarity, hybridization will occur.  The DNA probe is a singe-stranded molecule, conveniently labelled, using any standard method (e.g. a radioisotope), and hybridize with the target DNA, which is stuck to the membrane.
  • 12. Continuous  Thus the DNA probe binds with the sequences that are complementary to it among the thousands or millions of undetected fragments that migrate through the gel.  Desired fragments maybe detected after simultaneous exposure of the hybridized probe to X-rays, which on exposure appear as black spots on the X-ray film.
  • 13.
  • 14. Applications of RFLP  The important applications of RFLP are: 1. RFLP can be used paternity cases or criminal cases to determine the source of a DNA sample (i.e. it has forensic applications). 2. RFLP can be used to detect mutations.
  • 15. Advantages of RFLP  Some of the advantages of RFLP are as follow: 1. It is a simple process. 2. It is an accurate process. 3. No sequence information required.
  • 16. Disadvantages of RFLP  Some of the disadvantages of RFLP are as follow: 1. It is an expensive process. 2. It is slow and time consuming process. 3. Labour intensive. 4. Requires very large amount of DNA. 5. It is difficult to automate.