The document provides an overview of techniques used for assessing genetic changes, including DNA separation techniques like gel electrophoresis, capillary electrophoresis, and gradient centrifugation. It also discusses techniques for DNA analysis such as DNA sequencing, nucleic acid hybridization, DNA cloning, polymerase chain reaction (PCR), and DNA microarrays. PCR has advantages like being able to start from a single cell and generate products quickly, but requires knowledge of the target sequence. Real-time PCR allows quantifying DNA or cDNA. DNA microarrays detect mRNA or cDNA and have applications in gene expression analysis.

1. GOOD MORNING

2. TECHNIQUES OF ASSESSMENT
OF GENETIC CHANGES -
SARANYA ROY

3. CONTENTS
• INTRODUCTION
• HISTORY
• STRUCTURE OF DNA
• DNA SEPARATION TECHNIQUES
• TECH FOR DNA ANALYSIS
• DNA SEQUENCING
• POLYMERASE CHAIN REACTION
• REFERENCES

4. Meet the Gene Machine
What is a gene?
• A part of the DNA that codes
for a protein.
• Not all the DNA codes for
proteins.
• 30,000 genes in the human
genome.

5. Charles Darwin : -
In 1859 published ‘The Origin of Species‘
which expressed that living things appear to
be designed, but may actually be the result
of natural selection.
Darwin showed that living creatures evolved
over several generations through a series of
small changes.
Search for Genetic Material

6. Search for Genetic Material
• Gregor Mendel (1866):
• He found that ‘Factors’ determine the
characteristics a living thing will express.

7. Search for Genetic Material
• Freidrich Miescher(1868):
• 1st isolated a substance from
the nucleus of cells that he
called “nuclein.”

8. Search for Genetic Material
• Fredrick Griffith (1928):
• Studied effects of virulent
bacteria & nonvirulent bacteria
injected into mice
• Inserted foreign DNA and changed
protein/ trait - transformation
•believed that the transforming
agent was an inheritance
molecule.

9. Oswald Avery, Colin
MacLeod, & Maclyn
McCarty (1944):
Reported that “transforming
agent” in Griffith's experiment
was DNA.
Search for Genetic Material

10. Discovering the Structure
of DNA
• Phoebus Levine (1929):
• At the Rockefeller center, identified
the four bases of DNA.

11. Discovering the Structure of
DNAEdwin Chargaff (1950)
•Discovered a 1:1 ratio of adenine to thymine and guanine to
cytosine in DNA samples from a variety of organisms.

12. Discovering the structure of
DNA
Rosalind Franklin
(1952)
•Obtained sharp X-ray diffraction
photographs of DNA (Photo 51)
•Watson and Crick used her data 
revealed its helical shape

13. Discovering the structure of DNA
James Watson & Francis Crick
(1953)
•Discovered double helix
structure

14. STRUCTURE OF DNA

16. TECHNIQUES

17. DNA SEPARATION TECHNIQUES
WHY?..
• The individual pieces of DNA are in the order of megabases
in length – physically too large to be manipulated.
• large original chromosome DNA molecule → smaller
manageable pieces
• isolation & selection of the particular fragment is done.

18. • Mechanical shearing of chromosomes – produces breakages randomly to
produce a uniform size distribution of assorted molecules.
• Restriction endonucleases- enzyme employed to cut large DNA molecules
into defined shorter segments in a way that is reproducible.
• Cleave at specific sites – restriction sites
• Many earlier lab tech developed for separating & concentrating DNA
molecules based on size.

19. DNA SEPARATION TECHNIQUES
GEL ELECTROPHORESIS
CAPILLARY ELECTROPHORESIS
GRADIENT CENTRIFUGATION

20. GEL ELECTROPHORESIS
• Method of separation of macromolecules (DNA, RNA, Proteins) &
their fragments based on their size & charge.
• Neutral agarose gel electrophoresis – most basic type of gel
• Preparation of agarose gels of a specific % of agarose by mass(0.8-3%)
– creates a molecular sieve, with a mesh pore size being determined
by the % of agarose ( higher % - smaller pores)

21. Gels poured in a molten state into a rectangular container,
with discrete well formation at one end of the product.

23. Agarose powder + electrophoresis buffer GEL
Heated to a high temp. until all the powder has dissolved
Molten gel – poured in casting tray
“Comb” placed – wells
Gel solidified – comb removed – placed in tank
Buffer conducts electric current
Dye + DNA sample – moves from –ve to +ve charge
Visualisation of the DNA
Gel electrophoresis – Procedure…

24. Gel electrophoresis…
• Smaller fragments – move furthest from the wells
Larger fragments – move least from the wells
• All fragments of a given size will move at about the same
rate
- population of equal size molecules are concentrated
into discrete band at the same distance from the well.

25. • DNA-binding fluorescent
dye - ethidium bromide /
SYBR green added to the
gel
• the DNA bands are
stained, can be directly
seen when gel is exposed
to fluorescence-exciting
light.

26. Gel electrophoresis…
• A standard sample consisting of a set of
DNA molecules of a known size can be run
in one of the wells
• with samples to be estimated in other wells
and compared.

27. Disadvantages
•Labor intensive
•Bubbles in the gel - waste time and materials
•Acrylamide is a neurotoxin – Therefore dangerous to work with
•Have to be careful when loading – Cannot spill sample or load into wrong
lane
• Too much of heat – cause gel to “smile” – bands curve upwards at the
ends

28. • Analyzes the presence and behaviors of certain proteins - multiple
sclerosis, kidney disease and some cancers
• Testing the purity of an antibiotics
• Useful in production of vaccines
Applications

29. CAPILLARY ELECTROPHORESIS
• The gel matrix – fine capillary (25 to 100 µm in internal dia & 20-100 cm
length).
• A voltage of about 10-30 kV is applied across a narrow bore capillary.
• The capillary is filled with electrolyte soln --- conducts electricity through it.
• Capillary sucks sample up and through the polymer matrix based on high
voltage
• Buffer held at beginning and end of capillary –also sucked through polymer
• Larger DNA molecules are retarded by the polymer chains –travel slower
through capillary than smaller DNA molecules.
• A laser scanner reads the “bands” as they travel past

32. Capillary electrophoresis…
• High surface/volume ratio – efficient dissipation of the heat generated.
• Less sample is used.
• Allows the use of high electric fields
• Since high voltage are used – separation times are ↓.(min instead of hrs)
• Resolution & sensitivity is ↑.
• Capillary can be run clean of sample & reused
Advantages:

33. Capillary electrophoresis…
• Unlike the glass-supported slab gel (multiple lanes can be run
side by side) – capillary can handle only one sample at a time.
However, instruments with multiple parallel capillaries –
parallel analysis of multiple samples – further ↑ throughput.
• Instrument equipment costlier.
Disadvantages:

35. GRADIENT CENTRIFUGATION
• Process that uses centrifugal forces to separate & purify mixture of biological particle
in a liquid medium
• The 1st analytical ultracentrifuge was developed by Svedberg in 1920
Principle:
• Gravity & generation of centrifugal force to sediment different fractions
• Isopycnic banding – specific DNA molecules have unique densities based on G-C
content

36. GRADIENT CENTRIFUGATION
What for ?
• separating DNA molecules from other contaminating biomolecules
• For fractionation of specific small DNA from other DNAs
• Separate RNA from DNACells
• Isolate macromolecules such as proteins & nucleic acids

37. Gradient centrifugation…
• Procedure for separating particles (viruses, RNA, DNA) in which sample is
placed on a preformed gradient such as sucrose or caesium chloride (stable
density gradient)
• Macromolecules are “banded” in the gradient & can be collected as a pure
fraction.
• low density - near top of tube/ center of rotor
• high density - near bottom of tube or outside of rotor

38. Gradient centrifugation…
• When sample is placed on top of this gradient (or even mixed
uniformly within the gradient)
• subjected to continued centrifugation
• individual DNA molecules will migrate to a position in the
gradient where their density matches that of the surrounding
medium.

41. Gradient centrifugation…
Application:
Large-scale preparative purification tech
Isolation of DNA, RNA, proteins or lipids
Separation of antibodies & viruses
Remove cellular elements from blood – cell free serum or plasma
Use in hematology lab for PCV determination

42. TECH FOR DNA ANALYSIS

43. DNA SEQUENCING
NUCLEIC ACID HYBRIDISATION
DNA CLONING

44. • Determination of nucleotide sequences of DNA molecules in a particular
genome
• Extremely useful in
Determining the bp sequences of a part of or an entire gene
Analysing the function of a particular gene
Detecting a specific mutation
Detecting a gene’s degree of similarity with other known DNA sequences
DNA Sequencing

45. • Two methods:
Sanger’s dideoxy chain termination method
Chemical degradation method

46. Principle : NUCLEIC ACID HYBRIDIZATION
Blotting methods
DNA microarrays

47. NA PROBES
• ss DNA sequences radioactively / non-radioactively labeled
• Used to detect DNA / RNA fragments with sequence complementary to it.
• DNA probe can be labelled –
Isotopic – ³²P
Nonisotopic methods – modified nucleotides containing fluorophores
(fluorescein or rhodamine)

49. NUCLEIC ACID HYBRIDIZATION
• Hybridization – process of forming a double-stranded DNA molecule
between a single-stranded DNA probe and a single-stranded target
patient DNA.

50. BLOTTING METHODS
SOUTHERN BLOTTING
NORTHERN BLOTTING

51. SOUTHERN BLOTTING
• The technique was developed by E.M. Southern in 1975.
• It is used to detect the presence of a particular piece of DNA
in a sample.
• The DNA detected can be a single gene, or
it can be part of a larger piece of DNA such
as a viral genome.

52. Procedure ...
1.Restriction digest
2.Gel electrophoresis
3.Denaturation of the DNA
4.Southern transfer
5.DNA hybridization
6.Detection of probe by autoradiography

53. DNA Sample DNA Fragments
Gel electrophoresis
ds DNA ss DNA
Southern transfer Membrane (Southern blot)
Gel blotted for several hours
DNA fragments attach to membrane adhere firmly
Procedure ….
Restriction enz.
Baked at 80°C/
exposed to UV
radiation

54. DNA Fragments hybridized with radioactive probe
Base pairing
Bands detected – Autoradiograms
Procedure ….

56. Southern Blotting Procedure …
Visualization –
isotope – expose the membrane to film / phosphor image screen
Chemical labelling – chemiluminescent or fluorescent detection
The observed band intensity is related to the amt of
target on the membrane – quantitative method

57. Applications
• Determining viral copy number in a host cell sample
• Used to identify mutations, deletions & gene rearrangements
• In detecting restriction fragment length polymorphisms (RFLP)
• Used in prenatal diagnosis of genetic diseases such as sickle cell anemia
• Prognosis of cancer
• Diagnosis of HIV1 & infectious diseases

58. Disadvantages
• Takes a very long time – 3 to 4 days
• Technique sensitive
• Have to handle with harmful materials such as – radioactive
substances & ethidium bromide

59. NORTHERN BLOTTING
• The technique was developed by James Alwine & George Stark in 1977
• Technique for detecting specific RNAs separated by electrophoresis, by
hybridization to a labeled DNA probe.
• RNA cannot bind to cellulose nitrate – membrane amino benzyloxymethyl
filter paper
• Northern blotting takes its name from its similarity to the first blotting
technique, the Southern blot.

60. Prepare RNA samples – gel electrophoresis
Northern transfer
Probe preparation
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
Isotope
Non-isotope
Procedure

61. Applications:
• detection of RNA size
• the quality & quantity of RNA can be measured on the gel prior to
blotting – photographing of the gel.
• widely used to find gene expression & regulation of specific genes
• over expression of oncogenes and down regulation of tumor-
suppressor genes in cancerous cells

62. Advantages:
• detect small changes in gene expression that microarrays cannot.
• membranes can be stored & reprobed years after blotting.
Disadvantages:
• The chemicals used - can be a risk to the researcher, since
formaldehyde, radioactive material; ethidium bromide, and UV
light are all harmful under certain exposures.

63. • Evolved from Southern blotting
• The use of microarray for gene expression profiling was first reported in
1995
• It is a computer based mutation analysis method
• Made by placing oligonucleotides on a glass slide
• The word “array” means an orderly series, arrangement/ sequence
• 1 sq cm – several million nucleotides
• Detects mRNA or cDNA
DNA MICROARRAYS

64. • Principle is based on the fact that complementary sequences of DNA
can be used to hybridise, immobilised DNA molecules.
• The 4 major steps are:
DNA MICROARRAYS – Principles
Sample
preparation
& labelling
Hybridisation Washing
Image
acquisition &
Data analysis

66. DNA MICROARRAYS – APPLICATIONS

67. POLYMERASE CHAIN REACTION
• 1st developed by Kary Mullis in
1980s
• PCR is an in vitro technique for the
amplification of a region of DNA
which lies between two regions of
known sequence.
Nobel prize in chemistry1993

68. Principle:
• The PCR technique copies the target DNA by performing
repeated cycles each containing the following three main
steps :

69. STEPS

70. PCR Advantages
• PCR can be done from any cellular source containing nuclei, including
less invasive samples – buccal scrapings
• DNA from a single cell also is enough to start the PCR
• Use of taqDNA polymerase (heat –stable) – generates PCR products in
a matter of hours
• Real time PCR machines have reduced the time < 1 hr

71. PCR Disadvantages
• DNA from a contaminating extraneous source, such as
desquamated skin from a lab worker, may also get amplified –
false +ve results.
• Requires knowledge of nucleotide sequence of the target DNA
• Reagents and equipments are costly

72. Types of PCR

73. Nested PCR
• Uses 2 sets of primers, involving a double process of
amplification.
• Increased sensitivity - small amts of the target are detected.
• The 1st set of primers allows a 1st amplification.
• Product → 2nd PCR – 2nd set of primers.
• Prevents non-specific binding of primers & it’s amplification.

74. Multiplex PCR
• Allows simultaneous amplification of many sequences.
• It can detect different pathogens in a single sample
• This is achieved when in a single tube include sets of specific
primers for different targets.
• These primers are designed in such a way that they adhere to the
specific DNA sequences at similar temp.

75. Reverse Transcriptase PCR (RT-PCR)
• Designed to amplify RNA sequences (esp mRNA) through synthesis of
cDNA by reverse transcriptase (RT).
• This cDNA is amplified using PCR.
• Diagnosis of RNA viruses & evaluation of antimicrobial therapy .
• To study gene expression in vitro, as the cDNA retains the original
RNA sequence.

76. Challenges:
• Sample of mRNA is difficult to handle
• low stability at room temperature
• sensitive to action of ribonucleases & pH change

77. Real time PCR (qPCR)
• Quantify DNA or cDNA, determining gene or transcript numbers
present within different samples
• uses fluorescence detection systems – 2 types
intercalating agents
fluorophores

78. Real time PCR
The most commonly used probes -
• TaqMan probes
• molecular beacons probes, and
• FRET (fluorescent resonance energy transfer).

79. • Advantages :
• Faster result
• Reduced risk of contamination
• Ease in handling technology
• Process monitored in real time

80. Applications of PCR
Diagnosis Studying evolution
Forensic
Diagnosis of infectious
microbes that cause
maxillofacial infections

81. Studying fossils
Creating mutation
Antibiotic resistance
& multi-drug
resistance

82. REFERENCES
• EMERY`S ELEMENTS OF MEDICAL GENETICS 13TH EDITION - Peter Turnpenny, Sian
Ellard
• Lewin`s GENES 10 th edition
• GENETICS – ANALYSIS OF GENES & GENOMES 8TH EDITION – Daniel L Hartl; Maryellen
Ruvolo
• Polymerase Chain Reaction:Types, Utilities and Limitations - Patricia Hernández-
Rodríguez, Arlen Gomez Ramirez
• The application of DNA microarrays in gene expression analysis - N.L.W. Van Hal et al.
: Journal of Biotechnology 78 (2000) 271–280

83. REFERENCES CONTD..
• SOUTHERN BLOTTING - ED SOUTHERN - NATURE PROTOCOLS VOL.1 NO.2 2006
• DNA MICROARRAY TECHNOLOGY -WHAT IS IT AND HOW IS IT USEFUL? MIT –
Mandana Sassanfar and Graham Walker - Biology Science Outreach
• Amplification refractory mutation system (arms) and reverse hybridization in
the detection of beta-thalassemia mutations .Archives of Iranian Medicine, Vol
4, No 4, October 2001
• ARMS – PCR as an alternative, cost effective method for detection of fecB
genotype in sheep Indian Journal Of Biotechnology, July 2012.