This document discusses various techniques used in DNA analysis, including blotting techniques like Southern blot, Northern blot, and Western blot. It describes restriction fragment length polymorphism (RFLP) and how it can be used to detect genetic defects. It also explains polymerase chain reaction (PCR), a technique that amplifies specific DNA sequences, allowing them to be analyzed. PCR involves denaturing DNA, annealing primers, and extending new strands in repeated cycles. Applications of PCR include clinical diagnosis, DNA sequencing, and forensic medicine. Overall, the document provides an overview of key analytical techniques used to study DNA, such as blotting, RFLP, and PCR.

1. DNA analysisDNA analysis
Prof. Dr. V. P. AcharyaProf. Dr. V. P. Acharya
MBBS, MD, PhDMBBS, MD, PhD

2. • Can take many approaches from simple
quantification to complete sequence
• Common forms of analysis are
– size of DNA - electrophoresis
– homology of DNA - blotting, PCR
– arrangement of DNA - blotting, PCR,
sequencing

3. Blot transfer techniquesBlot transfer techniques
• Southern blot– DNA
• Northern blot– RNA
• Western blot– Protein
• Technique to visualise specific DNA, RNA
and Protein among other molecules

4. Southern BlotSouthern Blot
• E. M. Southern– 1976
• Useful for quantitating a specific gene
• Know mutation in the gene

5. • DNA extracted
• Digested with restriction endonucleases
• Fragments separated by gel
electrophoresis
• After a suitable time, DNA denatured by
mild alkali to ssDNA
• DNA transferred to nitrocellulose
membrane (Blot)
• Paper exposed to cDNA probes
• Paper exposed to X-ray– specific bands
seen

6. Application of Southern blotApplication of Southern blot
• Mutant genes detected e.g. HbS, Cystic
fibrosis, PKU, Viral DNA
• Forensic purpose
• RFLP for pathological disease
• Confirmation of DNA cloning results

7. Northern BlotNorthern Blot
• Size and quantitate specific RNA molecule
• Alkali not used
• cDNA probes are used- RNA-DNA
Hybridisation
• Similar to Southern blot
Application:
Detection of gene expression in a tissue

8. Western BlotWestern Blot
• Size and quantitate specific protein
molecule
• No complementary substance required
Application:
Detection of HIV viral proteins- confirmatory
test

9. Dot blot/ Slot blot techniqueDot blot/ Slot blot technique
• Modified Western blot technique
• No electrophoresis- sample directly
applied as a dot on the blot paper and
analysed
• Can quantify DNA, RNA or specific
proteins

10. Restriction Fragment length PolymorphismRestriction Fragment length Polymorphism
• Variation in DNA sequence–
Polymorphism
• Occurs once every 500 nucleotides &107
times per genome
• Normally takes place in non-coding
regions
• These variations cause variation in the
restriction sites and thus length of
restriction fragments
• Inherited difference in the pattern of restriction sites- RFLP

11. Application of RFLPApplication of RFLP
• Detects genetic defects in parents and
fetal tissues e.g. sickle cell anaemia,
Retinoblastoma, Alzeihmer’s disease
• Forensic medicine– DNA fingerprinting

12. VNTR (Variable number tandem repeats)VNTR (Variable number tandem repeats)
• Unique to each one
• Minisatellites
• Different numbers of base sequences
between 2 points of DNA
• DNA fingerprinting
VNTR (D1S80) in 6 individuals

13. Microsatellites/ Simple tandemMicrosatellites/ Simple tandem
repeats (STR)repeats (STR)
• 2-3 nucleotides
• Evenly distributed throughout the genome
• More preferred over VNTR
• Can be identified by PCR

14. DNA SequencingDNA Sequencing
• Maxam Gilbert technique
• Sanger’s method-
interrupted enzymatic
cleavage
• Automated DNA sequencing
• DNA chips- Microarray

15. P C R

16. Polymerase Chain reaCtionPolymerase Chain reaCtion
• Karry Mullis– 1994
• Works like a photocopier
• PCR is a technique that takes a specific
sequence of DNA of small amounts and
amplifies it to be used for further testing.

17. PCR targetsPCR targets
The targets in PCR are the sequences of DNA
on each end of the region of interest, which can
be a complete gene or small sequence.
The number of bases in the targets can vary.
TTAAGGCTCGA . . . . AATTGGTTAA
The . . . . Represents the middle DNA sequence,
and does not have to be known to replicate it.

18. Requisites for PCRRequisites for PCR
• DNA nucleotides, the building blocks for the new
DNA
• Template DNA, the DNA sequence that you
want to amplify
• Primers, single-stranded DNAs between 20 and
50 nucleotides long (oligonucleotides) that are
complementary to a short region on either side
of the template DNA
• DNA polymerase (Taq Polymerase, Tth
polymerase), a heat stable enzyme that drives,
or catalyzes, the synthesis of new DNA

19. Steps of PCRSteps of PCR
• Denaturation—940
c
• Annealing—540
c
• Extension-- 72°C
• 20-40 cycles, Thermocycler

21. Exponential increase in the no. of copies of DNAExponential increase in the no. of copies of DNA

22. 3 stages in each cycle of PCR3 stages in each cycle of PCR

23. It can’t go on infinitelyIt can’t go on infinitely
• Saturation
• Competition for primers
• Rapid cycling- less non-
specific products
• Size matters- primer
length ideal between 18-
25bp
• Small length DNA should
be chosen to be copied

24. Variations of PCRVariations of PCR
• RT-PCR
• Nested PCR
• Real-time PCR
• Multiplex PCR

25. Applications of PCRApplications of PCR
• Clinical diagnosis
 Prenatal diagnosis
 Retroviral infections
 Bacterial infections
 cancers
• DNA sequencing
• Comparative studies of genome
• Forensic medicine

26. Other forms of target amplificationsOther forms of target amplifications
• Transcription-based amplification method
• Strand displacement amplification
• Loop-mediated amplification
• Whole genome and whole transcriptome
amplification

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