Molecular Technique.pdf MLT students Ind

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1. MOLECULAR AMPLIFICATION TECHNIQUES
Nucleic acid (NA) amplification methods fall into 3 categories:
•Target amplification systems
•Probe amplification systems
•Signal amplification

2. Target Amplification Methods
•PCR – POLYMERASE CHAIN REACTION
•NASBA - Nucleic Acid Sequence-Based Amplification
•TMA – Transcription Mediated Amplification
•SDA - Strand Displacement Amplification
Signal Amplification Methods
•bDNA – Branched DNA probes
Probe Amplification Methods
•LCR – Ligase Chain Reaction

3. PCR Techniques

4. ➢ PCR is process of amplification of specific DNA sequences (a segment of DNA that
lies between 2 regions of known sequence).
➢PCRis an exponentially progressing synthesis of the defined target DNA sequences in
vitro.
➢PCR can make billions of copies of a target sequence of DNA in a few hours.
-It can be thought of as:a Molecular Photocopier
Polymerase Chain Reaction (PCR)

5. Essential components of PCR
1)DNA template: (DNA that contains the target sequence to copy it)
2)Thermostable DNA Polymerase (e,g. Taq DNA Polymerase )
3)All 4 nucleotide triphosphates (d NTP’s) : to make new DNA
4)Buffers: KCL, Tris-HCl pH buffer(8.3-8.8) and Bovine Serum Albumin.
Contains MgCL
2 (Mg++) as cofactor for DNA Polymerase
5) Two primers (forward and reverse primers): Two short, single-stranded DNA
molecules (usually 16-30 nucleotides)
6) Thermocycler: device that can change temperatures dramatically in a very short
period of time

6. Thermocycler
Thin walled
tubes

7. Taq DNA Polymerase
•PCR involves very high temperatures (94 C to denature DNA).
•Most DNA polymerases would denature and become inactivated at the high temperatures
of PCR.
•Taq DNA polymerase is thermostable enzyme (resist to denaturation by heat)
•Taq DNA polymerase was purified from the hot springs bacterium Thermus aquaticus in
1976
•Taq DNA polymerase has maximal enzymatic activity at 75 C to 80 C, and substantially
reduced activities at lower temperatures.

8. The Basics of PCR Cycling
PCR process is carried out in cycles. Each cycle of PCR have 3
steps such as denaturation, annealing and extension.

9. The three main steps of PCR
•In a PCR reaction, the following series of steps is repeated 20-40 times
Step Temp.
DenaturationAt 94C the DNA is denatured (the two strands are separated)
AnnealingAt 40C- 65Cthe primers anneal (or bind to) their complementary sequences
on the single strands of DNA
ExtensionAt 72C DNA Polymerase extends the DNA chain by adding nucleotides
to the 3’ ends of the primers.

10. R e a l
-

T I m e P C R
Real
-
Time PCR
DNA
Nucleotides
DNA Polymerase
Forward Primer
Reverse Primer
PCR

11. Real
-
Time PCR
25°C95°CThermal Cycle 65°C
DenaturationAnnealing
R e a l
-

T I m e P C R

12. Extension
72°C65°CThermal Cycle
Annealing
Real
-
Time PCR
R e a l
-

T I m e P C R

13. R e a l
-

T I m e P C R
Real
-
Time PCR

14. R e a l
-

T I m e P C R
Real
-
Time PCR
36 cycles amplifies the DNA fragment of
interest 2
36
(68 billions) copies of DNA!

15. R e a l
-

T I m e P C R
Real
-
Time PCR
Agrose Gel
PCR
Mixing with Loading Dye
Loading the samples into the gel

16. Detection of amplified DNA :
a. Staining of amplified DNA product with ethidium bromide during agarose gel
electrophoresis.
b. Labelling of primers (DNA probe) with fluorophores before PCR amplification.

17. Primers:
•Designing appropriate primers is essential to the successful outcome of a PCR
experiment.
•When designing a set of primers to a specific region of DNA desired for
amplification,
✓one primer should anneal to the plus strand, which by convention is oriented in
the 5' → 3' direction (also known as the sense or non-template strand)
✓and the other primer should complement the minus strand, which is oriented in
the 3' → 5' direction (antisense or template strand).

18. Characteristics that should be considered when designing primers.
1.Primer length should be 15-30 nucleotide residues (bases).
2.Optimal G-C content should range between 40-60%.
3.The 3' end of primers should contain a G or C in order to clamp the primer and
increasing priming efficiency. Avoid placing morethan three G or C nucleotides at
the 3’-end to lower the risk of nonspecific priming .
4.The 3' ends of a primer set, should not be complementary to each other (to
avoid primer dimers formation), nor can the 3' end of a single primer be
complementary to other sequences in the same primer (to avoid hairpin loop
structures formation) .
.

19. Primer Examples
•Primer 1: AGCGGGGGATGGGGCC
•Primer 2: GTCATATATATATCATATTC
•Primer 3: GTCTACGTCAGTGCATCA
•Primer 1: high GC content & repeats of 5 Gs
•Primer 2: low GC content & runs of 5 Ats
•Primer 3: optimum GC content , No repeats, No runs.

20. Primer Examples
5’ 3’
5’
5’
5’
3’
3’
3’

21. 5. Melting Temperature (Tm): Temperature at which 50% of the DNA is double stranded
and 50% is single stranded. Optimal Tm for primers range between 52-58 °C, although the
range can be expanded to 45-65 °C. The final Tm for both primers should differ by no more
than 5 °C.
•for each primer calculate Tm = 4(G + C) + 2(A + T)
Oc
6. Annealing temperature:
temperature set about 5°Cbelow the T
m of the primers (ideally between 52 °C to 58°C).
T. anneal= Tm primer- 5° C.
For example: primer forward Tm=57
0
C and primer reverse Tm=59
0
C --→we can select 52
0
C
as annealing temperature
•Decrease in annealing temperature result in non-specific binding
•Increase in annealing temperature result in reduced yield
if the annealing temperature is too high, primers are unable to bind to the template.
➢The distance between the primer binding sites will determine the length of the PCR product (amplicon).
➢Then the length of amplicon used to adjust time of extension
➢1Kb need 1 min for Taq polymerase.

22. Large (Klenow) Fragment of E. coli DNA Polymerase I
The large or Klenow fragment of DNA polymerase I has DNA polymerase (to synthesize
complementary strands during DNA replication) and 3' -> 5' exonuclease activities (proof
reading), and is widely used in molecular biology.

24. The exo- Klenow fragment of E. coli DNA Polymerase I
•the 3' → 5' exonuclease activity of Klenow fragment can also be undesirable for
certain applications.
•This problem can be overcome by introducing mutations in the gene that encodes
Klenow. This results in forms of the enzyme being expressed that retain
5' → 3' polymerase activity, but lack any exonuclease activity (5' → 3' or 3' → 5').
•This form of the enzyme is called the exo- Klenow fragment.
•The exo-Klenow fragment is used in some fluorescent labeling reactions for
microarray.

25. Taq DNA polymerase
✓It has DNA polymerase activity
✓It has a 5'-3' exonuclease activity
✓It lacks 3'-5' exonuclease activities (proof reading), that may result in a
high error rate (mutations per nucleotide per cycle) which affects the
fidelity of the PCR , causing accumulation of a large proportion of amplified
DNA with incorrect sequence in the final product.
❑Several "high-fidelity" DNA polymerases, with 3' to 5' exonuclease activity.
Examples include: KOD DNA polymerase, Vent, Pfu DNA polymerase, and
Pwo,

26. Thermostable Polymerases
Alvin Submersible for Exploration of Deep Sea Vents

27. Some Applications of PCR
•Molecular Research and
Biotechnology
1) The Human Genome Project
2) Evolutionary studies
3) Analyse gene expression by
measuring RNA levels
4) Detect presence of introduced
gene (transgene)
•Forensics
1) Identify criminal suspects
2) Paternity cases
•Medical Diagnostics
1) Diagnosis and characterisation of
Infectious diseases:
-Detect presence of viral pathogens (HIV,
hepatitis B)
-Detect presence of pathogenic bacteria
(Chlamydia trichomatous , Mycobacterium
tuberculosis, Ureaplasma urealyticum, Mycoplasma
spp)
2) Diagnosis and characterisation of human
genetic diseases (Huntington's disease)
3) Diagnosis and characterisation of
Neoplasia(Cancer)
4)In therapy resistant assessment

28. Different Types of PCR Techniques
•1.Conventional PCR
•2. Nested PCR
•3. Multiplex PCR
•4. RT-PCR
•4. Touchdown PCR
•5. Inverse PCR
•6. Allele specific PCR
•7. Asymmetric PCR
•8. Arbitrary PCR
•9. Core sample PCR
•10. Degenerate PCR
•11. Assembly PCR
•12. Dial-out PCR
•13. Digital PCR
•14. Traditional PCR
•15. Hot start PCR
•16. In-silico PCR
•17. Inter sequence PCR
•18. Ligation-mediated
PCR
•19. Methylation-
specific PCR
•20. Miniprimer PCR
•21. Nano particle PCR

•22. Overlap-extension PCR
•23. Real-time or Quantitative PCR
•24. Solid phase PCR
•25. Suicide PCR
•26. Thermal asymmetric
interplaced PCR
•27. Semiquantative PCR
•28. Colony PCR
•29.. After exponentional PCR
•30. Standard PCR
•31. Qualitative PCR

29. 1. Nested PCR
•Two sets of primers are used to amplify a single region
of DNA.
•The first set is an amplified sequence and
•the second set is complementary to the first sequence
which will be shorter than the first amplified product.
• Nested PCR is used to reduce the contaminations in
products due to the amplification of unexpected primer
binding sites.
•Nested PCR allowing for very low probability of
nonspecific amplification.

30. 2.Multiplex PCR
•Use of multiple sets of primers in one reaction to detect more than one organism or
to detect multiple genes in one organism.

31. 3.Hot-Start PCR
•The major obstacle to routine, sensitive and specific PCR amplification is
amplification of non-target sequences in background DNA (mispriming) and
primer-oligomerization.
•This mispriming and primer-oligomerization occurs mainly during pre-PCR setup
when all reactants have been mixed, usually at room temperature, before PCR
thermal cycling is started.
•There are several methods which allow the DNA polymerase to remain inactive until the
initial denaturation period has completed, including the use of a solid wax barrier, anti-DNA
polymerase antibodies, and accessory proteins.
•Platinum Taq , AmpliTaq Gold or HotStar Taq DNA Polymerase is provided in an inactive
form which is then heat activated.
•Initial temperature at 95c for 1-3 min

33. final extension time is recommended to allows synthesis of many uncompleted amplicons to finish
Standard 3-step PCR Cycling
Cycle step Temperature Time
Number of
Cycles
Initial Denaturation94 °C to 98 °C 1-3 minutes 1
Denaturation
Annealing
Extension
94 °C
5 °C below T
m
70 °C to 80 °C
10 to 60 seconds
30 seconds
Amplicon and DNA polymerase
dependent
25-35
Final Extension 70 °C to 80 °C 5 minutes 1
Hold* 4 °C ∞ 1

34. Reverse Transcription- Polymerase chain reaction (RT-PCR)

RT-PCR, one of the most sensitive methods for the detection and analysis of RNA.
RNA cannot serve as a template for PCR, So RNA must be transcribed into complementary
DNA.
Reverse transcriptase is a enzyme that functions as a RNA-dependent DNA polymerase.
They are encoded by retroviruses, where they copy the viral RNA genome into DNA prior to its
integration into host cells. In the laboratory, it is used to convert RNA to cDNA by reverse
transcription.
Reverse transcriptase Examples:
✓M-MuLV RT from Moloney murine leukemia virus or AMV RT from Avian myeloblastosis
virus, Viral RTs, used mainly in two-step assays,
✓rTth DNA Polymerase from Thermus thermophilus (bacteria), functions as both a reverse
transcriptase and DNA polymerase , used in a single-tube.

35. Reverse transcriptases have two activities:
• DNA polymerase activity: Synthesis of cDNA using primers
• RNase H activity: Digestion of cDNA:RNA heteroduplex
RT-PCR involves two processes:
1. RT – Reverse Transcription
During this step converting RNA to cDNA by reverse transcriptase
we synthesize single stranded DNA from RNA template
2. PCR – Polymerase chain reaction
Using gene-specific primers we amplify a certain part of our gene of interest to get
enough amount for further analysis

36. 36
How does it work ?

37. Depending on the requirement, the primer for
first cDNA strand synthesis can be of 3 types;
1. Oligo dT (12-20 nts) : is used as universal
primer which binds to poly (A) tail of mRNA;
and
2. Reverse primers are specifically designed
which can hybridize to a particular target genes
or defined mRNA family.
3. Random hexamer primers : are used to
initiate transcripts from multiple points along
the RNA, including ribosomal RNA (rRNA), thus
producing more than one cDNA per original
target.

39. RT-PCR can be either :
a one-tube assay using a single buffer (with rTth DNA Polymerase), or
a two-tube assay ( with Viral RTs) where both first-strand cDNA synthesis and the
subsequent PCR step are performed separately under optimal conditions for the respective
polymerases.

40. Uses of RT-PCR
1.Gene Expression (The mRNA)
2.Detecting and Studying the RNA viruses.
3.Cloning of eukaryotic genes in prokaryotes

41. Gene Expression (looking for mRNA !!!)
AAAAA
AAAAA
AAAAA
mRNA
mRNA
mRNA
Reverse Transcription
cDNA analysis

42. Gene Expression (looking for mRNA !!!)
total RNA
tRNA
rRNA
mRNA
2.5-5%
AAAAA
Only mRNA has a poly-Adenin tail at the 3’ end
RNA isolation
•Most of the RNA is unimportant for us (tRNA, rRNA)
•mRNA population consists of about 35000 different kind
• Strong secondary structure – enzyme cannot work

43. AAAAA
AAAAA
AAAAA
TTTTT
TTTTT
TTTTT
TTTTT
Reverse
transcriptase
dATP
dCTP
dGTP
dTTP
RNase inhibitor
mRNA
mRNA
AAAAA
TTTTTmRNA
TTTTT
cDNA
37 ºC
RT
cDNA
RT
RT ready
43
Gene Expression (looking for mRNA !!!)
65 ºC
37 ºC
Denature
Anneal
+
Elongate
PCR

44. 44
total RNA + oligodT
37 ºC – 1 hour
anneal + elongate
65ºC – 10 min
Denature
(hair pain
structure of
mRNA)
Add:
Enzyme RT
dNTPs
RNasin
RT ready
RT:
PCR:
DNA pol
dNTPs
primers
Buffer
MgCl
2
95ºC
3 min
denature amplify
95ºC – 30 sec
55ºC – 30 sec
72ºC – 1 min
72ºC
10 min
finish
PCR ready
template
Gel analysis
30 cycles
Gene Expression (looking for mRNA !!!)

45. Thank you

46. Allelic Discrimination Using TaqMan Probes

47. PCR and Disease
•Primers can be created that will only bind and amplify
certain alleles of genes or mutations of genes
•This is the basis of genetic counseling and PCR is used as part of
the diagnostic tests for genetic diseases.
•Some diseases that can be diagnosed with the help of PCR:
•Huntington's disease
•cystic fibrosis
•Human immunodeficiency virus

48. Human Immunodeficiency Virus (HIV)
•HIV is a retrovirus that attacks the immune system.
•HIV tests rely on PCR with primers that will only amplify a
section of the viral DNA found in an infected individual’s
bodily fluids.
Therefore if there is a PCR product, the person is likely to be
HIV positive. If there is no PCR product the person is likely to be
HIV negative.
•Protein detection based tests are available as well but all US blood is
tested by PCR.

49. PCR and Forensic Science
•It is often of interest in forensic science to identify individuals genetically.
In these cases, one is interested in looking at variable regions of the
genome as opposed to highly-conserved genes.
•PCR can be used to amplify highly variable regions of the human
genome. These regions contain runs of short, repeated sequences
(known as variable number of tandem repeat (VNTR) sequences) . The
number of repeats can vary from 4-40 in different individuals.
•Primers are chosen that will amplify these repeated areas and the
genomic fragments generated give us a unique “genetic fingerprint” that
can be used to identify an individual.

50. PCR Applications to Forensic Science
•Paternity suits -Argentina’s Mothers of the plaza and their search
for abducted grandchildren
•Identifying badly decomposed bodies or when only body fragments
are found - World trade center, Bosnian , Iraq & Rwandan mass
graves

51. Applications of PCR
PCR can also be used in forensic testing.
The DNA sequences used are of short repeating patterns called VNTR
(variable number of tandem repeat), which can range from 4 to 40
nucleotides in different individuals.
One set of VNTR locus are inherited from the mother and one set from the
father.
The genes are amplified using PCR, and then run through electrophoresis.
The position of the two bands on the electrophoresis gel depends on the
exact number of repeats at the locus.
Three VNTR loci from suspects, along with the DNA from the scene are run
through PCR amplification, and then through electrophoresis.
This gives six bands, which can have common bands for some individuals,
but the overall pattern is distinctive for each person.

52. Applications of PCR

53. Applications of PCR

54. ❖Forensics : This gel is a copy of the data obtained by PCR of VNTR
loci in the labeled samples.
Applications

56. What are SNPs ?
•Single Nucleotide Polymorphisms
(point mutation is SNP at lower frequency)
•found about every 1.0 -1.5kb
A C G T T G G A T A C
T G C A A C C T A T G
5’ 3’
3’ 5’
A C G T T G T A T A C
T G C A A C A T A T G
5’ 3’
3’ 5’

57. For Allele 1 - only VIC™ dye signal is generated
SNP scoring assay
Using TaqMan MGB probes
C
G
A
C
NFQFAM
VIC
G
T
A
T
FAM
VIC
For Allele 2 - only FAM™ dye signal is generated
NFQ
NFQ
NFQ

58. For Allele 1 - only VIC™ dye signal is generated
For Allele 2 - only FAM™ dye signal is generated
SNP scoring assay
Using TaqMan MGB probes
G
C
A
C
NFQFAM
VIC
G
T
A
T
FAM
VIC
NFQ
NFQ
NFQ

59. wildtype Allele 1
Heterozygous Allele 1 / Allele 2
homozygous Allele 2
+
Genotype Allele FAM VIC
RR
R
SNP scoring assay
Using TaqMan MGB probes

60. •For a bi-allelic system, probes specific for each allele are included in
the PCR assay.
•The probes can be distinguished because they are labeled with
different fluorescent reporter dyes (FAM
TM
dye and VIC
TM
dye in the
next figure).
5`Nuclease and Allelic Discrimination

61. •A fully hybridized remains bound during strand displacement, resulting in
efficient probe cleavage and release of the reporter dye.
•A mismatch between probe and target greatly reduces the efficiency of
probe hybridization and cleavage.
5`Nuclease and Allelic Discrimination

62. •Thus, substantial increase in FAM or VIC fluorescence indicates
homozygosity for the FAM- or VIC-specific allele.
•An increase in both signals indicates heterozygosity.
5`Nuclease and Allelic Discrimination

63. Indicates…A substantial increase in..
Homozygosity for allele 1VIC
TM
fluorescence only
Homozygosity for allele 2FAM
TM
fluorescence only
Heterozygosity Both fluorescence
legend
VIC
FAM
Quencher
AmpliTaq
Allele
1
Allele
2
Match
mismatch
Mismatch
Match
Strategy of Allelic Discrimination Assay

64. Indicates…A substantial increase in..
Homozygosity for allele 1VIC
TM
fluorescence only
Homozygosity for allele 2FAM
TM
fluorescence only
Heterozygosity Both fluorescence
Discrimination of alleles for human SNP rs2589

65. What is Real-Time PCR used for?
Real-Time PCR has become a cornerstone of molecular
biology. Just some of the uses include:
•Gene expression analysis (RNAs)
•Cancer research
•Drug research
•Disease diagnosis and management
•Viral quantification
•Food testing
•Percent GMO (Genetically modified food)
•SNP detection
•Copy Number Variation (CNV)

66. Real-Time
PCR in
Gene
Expression
Analysis
Example: BRCA1 Expression Profiling
BRCA1 is a gene involved in tumor suppression.
BRCA1 controls the expression of other genes.
In order to monitor level of expression of BRCA1,
real-time PCR is used.
DNA
mRNA
Protein
BRCA1
Determine gene
expression and
publish scientific
paper!

67. Real-Time
PCR in
Disease
Manageme
nt
Example: HCV Treatment
Drug treatment for HCV infection often depends on
monitoring the “viral load”.
Real-Time PCR allows for direct measurement of the
amount of the virus RNA in the patient.
Viral RNA
Measure amount
of virus, adjust
prescriptions.

68. Real-Time
PCR in Food
Testing
Example: Determining percentage of GMO food
content
Determination of percent GMO food content
important for import / export regulations.
Labs use Real-Time PCR to measure amount of
transgenic versus wild-type DNA.
wt DNA
GMO DNA
International shipments
depend on results!

69. Real-Time PCR in SNP detection

70. Real-Time PCR in Copy Number Variation
(CNV)