Molecular diagnostic

technologies-1

Manipulation of nucleic acids

and amplification of genetic

material

ZAHRA HASAN, PhD

Associate Professor

Section of Molecular Pathology 1

Objectives of this class

• To relate the question (target genetic

material) with the assay required

• To understand how polymerase chain

reaction works

• To understand the methods for visualising

PCR products

• To understand conventional and real time

PCR

• To differentiate qualitative and quantitative

PCR 2

Amplification

• Why ?

• How ?

• What – material ?

• Which – protocol ?

3

Extraction of nucleic acid from

clinical specimen• Viral or bacterial sample

• Human genetic material

• Source of sample

– Blood / serum

– Body fluids:

– pulmonary (BAL, sputum)

– extrapulmonary specimen (ascites, CSF,

Pleural fluid, pus)4

Manipulation of DNA/RNA• Lysis of cell walls

– DNA/RNA release after cellular lysis using chemical (SDS) or enzymatic (Proteinase K, lysozyme)

• Extraction of DNA/RNA from nucleo-proteins– Use of prepared spin column

– Extraction using a salting out method

• Isolation / Precipitation of nucleic acids by Salting out and centrifugation

• Ethanol precipitation (EtOh increases

hydrophobicity)

• Salts increases ionic strength of solution (NaCl,

NH4Ac, LiCl)5

• Protection from nucleases

(DNAses – degraded at 95 C; RNAses are not degraded)

Store appropriately (temp, sterile)

-20 C for longer term storage, 4 C for short term (week)

Storage of DNA

6

Quantification of nucleic acids

Spectrophotometric measurement

based on Optical Density

Nucleic acids have maximum absorption at 260nm

Proteins have max absorption at 280 nm

Purity determined by DO 260/280 ratio

ODs of pure solution

DNA 50 mg/ml OD 260/280 = 1.8

RNA 40mg/ml OD260/280=2.0

7

Nucleic acid based amplification methods

PCR

Conventional – end point analysis

Real-time PCR – kinetic flourescence analysis

Qualitative

Quantitative

Nested- PCR

ARMS PCR

PCR –RFLP

Melting curve analysis for a) differentiation, b) SNP analysis

Ligase Chain Reaction

Reverse line probe assay

Transcription mediated amplification- hybrid capture

Strand displacement assay

8

Utility of PCR

• the detection and diagnosis of infectious

diseases

• the diagnosis of hereditary diseases

• For DNA cloning for DNA sequencing

• DNA-based phylogeny, or functional analysis of

genes;

• the identification of genetic fingerprints (used in

forensic sciences and paternity testing); and

• For determining concentration of target (viral

load; mutation load) 9

DNA replication

Strands complementary and anti-parallel

One strand built 5’ – 3’ direction

Other strand built 3’ – 5’ direction

Each strand can be template for other

Semi-conservative replication

Helicase

DNA polymerase

DNA ligase

primase

10

Requirements for PCR

• Primers designed to known sequence ‘flanking’

unknown region, 18-30 bp in size

• DNA polymerase from Thermophilus aquaticus

• dNTPs

• MgCl2

correct Tm

multiple cycles

Automated PCR cycler

CONTROLS, +ve and -ve

11

95C

72C

56C

12

Careful primer design is key!

• length of primers

– 18-25 bp for PCR assay

• Size of expected product depends on assay (50-150

bp for real time PCR)

• Specific and stringent

• high efficiency

• no primer-dimers

• Choice of polymerase – depends on PCR product

size and also the GC content of the target nucleic

acid sequence

• End of primer should be A or T and fewer GC strings

• Primers should have similar Tm (melting temp) 13

Tm of primers should be similar

14

Factors determining ‘Tm’: the

temperature at which 50% of all

sample is in a denatured form

• Strand length- determines H bonding

• Base composition- more bonds in GC,

>GC% requires higher temp.

• Chemical environment – Na+ stabilises

helix while urea and formamide destabilise

it

15

Example; selection of primers

for PCR of region of interest

16

Design based on the positive strand of DNA

Forward primer is 5 to 3 ‘ direction

Reverse primer is 3’ – 5 ‘ direction

5 ‘ ---------------------3’

3 ‘ ---------------------5’

RT-PCR

• RNA as starting material – reverse

transcription required

• RNA viruses -CCHF, HIV

• Human Gene expression – oncogenes

• Study of transcriptional responses

17

IMPORTANCE OF RNA

QUALITY• Should be free of protein (absorbance

260nm/280nm)

• Should be undegraded (28S/18S ~2:1)

• Should be free of DNA (DNAse treat)

• Should be free of PCR inhibitors

– Purification methods

– Clean-up methods

18

Storage of RNA• Store RNA at – 70 C

• RNAses are extremely stable and refold after denaturation

• Inhibit cellular RNAses

• Separate RNA from DNA and proteins

• Keep RNAse out

• RNAses can be degraded by Diethylpyrocarbonate (DEPC) /RNAse away

• Use RNAse inhibitor in reactions

19

EXON 1 EXON 2INTRON 2 DNA

EXON 1 EXON 2 RNA

20

RT-PCR based transcriptional analysis of genes

- looking at human gene expression; e.g. for oncological changes (Bcr gene)

- primers should be designed appropriately

Ideally should not give a DNA signal cross exon/exon boundary

OVERVIEW

RT-PCR

tissue

extract RNA

copy into cDNA

(reverse transciptase)

PCR

analyze results21

Importance of controls

• negative control (no DNA)

– checks reagents for contamination

• no reverse transcriptase control

– detects if signal from contaminating DNA

• positive control

– checks that reagents and primers work

– especially importance if trying to show absence of expression of a gene

22

Importance of cleanliness in

PCR• Contamination is major problem

• Huge amplification contributes to this

23

Electrophoresis - properties

• Nuc. Acids are charged mols and migrate

in the direction of the electrode with

opposite charge (anode)

• Electrophoretic mobility:

– dsDNA migrates inversely proportional to

log10 of bp

– Base composition, conc. of gel, composition

and ionic strength of buffer, temperature and

intercalating dyes

24

DNA separation

Agarose gel electrophoresis:

visualisation with ethidium bromide (UV illumination required)

SYBR green dye (fluorescence measurement)

Separation on agarose gels depends on

1. Size

2. Electrophoretic mobility

3. The agarose concentration

Smaller molecules travel more rapidly

Larger molecules migrate more slowly

Concentration of agarose is determined by size of product of interest

0.8% gel for products 1 – 10 kb

1% agarose for products 500bp to 2000bp

2% agarose for sizes 50 – 500 kb

NOTE

It is important to use DNA size ladders to run alongside samples on a

Gel in order to size the PCR products/DNA

26

How are you going to measure

the PCR product• Directly

– Ethidium bromide staining

– Sybr green dye

• Indirectly

– In addition to primers, add a fluorescently

labeled hybridization probe

– Second set of probes

27

Analysis of conventional PCR

based on agarose gel

1 2 3 4 5 6 +C -C

28

End point analysis

REAL TIME PCR

USING SYBR GREEN

29

REAL TIME PCR

• kinetic approach

• early stages

• while still linear

www.biorad.com30

31

www.biorad.com

2a. excitation

filters

2b. emission

filters

1. halogen

tungsten lamp

4. sample plate

3. intensifier5. ccd

detector

350,000

pixels

32

0

200000000

400000000

600000000

800000000

1000000000

1200000000

1400000000

1600000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBERA

MO

UN

T O

F D

NA

1

10

100

1000

10000

100000

1000000

10000000

100000000

1000000000

10000000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBER

AM

OU

NT

OF

DN

A

CYCLE NUMBER AMOUNT OF DNA

0 1

1 2

2 4

3 8

4 16

5 32

6 64

7 128

8 256

9 512

10 1,024

11 2,048

12 4,096

13 8,192

14 16,384

15 32,768

16 65,536

17 131,072

18 262,144

19 524,288

20 1,048,576

21 2,097,152

22 4,194,304

23 8,388,608

24 16,777,216

25 33,554,432

26 67,108,864

27 134,217,728

28 268,435,456

29 536,870,912

30 1,073,741,824

31 1,400,000,000

32 1,500,000,000

33 1,550,000,000

34 1,580,000,000

33

SERIES OF 10-FOLD DILUTIONS34

Importance of controls

• negative control

– checks reagents for contamination

35

Amplification

Qs. WHAT IS THE VALUE OF USING A PROBE IN THE

REAL-TIME PCR ASSAY?

Ans. The signal is not always specific

to the target sequence

36

Example; selection of primers

for PCR of region of interest

37

Design based on the positive strand of DNA

Forward primer is 5 to 3 ‘ direction

Reverse primer is 3’ – 5 ‘ direction

5 ‘ ---------------------3’

3 ‘ ---------------------5’

DNA probes on the principle of

Nucleic acid hybridisation

• Involves mixing of two sources of nucleic

acids, a probe which typically consists of a

homogenous population of identified

molecules (i.e, synthesized oligos) and a

target, which consists of a complex,

heterogenous population of nucleic acid

molecules

38

Nucleic acid probes detect a Unique Target

Sequence - RNA or DNA

Must be single stranded to hybridise to target

Short- 15-50 nucleotides

Labeled at 5’ or 3’ end:

Radioactive methods - P32, S35

Non-radioactive methods –

biotin-streptavidin, digoxygenin

Fluorescent labels; fluorescein, rhodamine, texasred

39

Fluorophore colour Excitation max Emission max.

Fluorescein green 494 523

Rhodamine red 555 580

40

Real-time PCR assays-1

TaqMan assay -A TaqMan probe has 5’ Reporter dye and a 3’ dye

Quencher

PRIMERS (F and R) bind

Probe binds upstream to PRIMERS

DNA pol will synthesize new strand,

Extend to Probe site

CLEAVE off the ®

The cleaved ® will flouresce

Fluorescence will be detected

41

Real-time PCR assays-2

FRET probe assays – Flourescence Resonance Energy Transfer

between a Donor and an Acceptor probe

PRIMERS (F and R) bind

PCR product generated

Probes bind to internal sequence of

PCR product

Probe 1- 5’ Red label/640nm – with 3’

Phosphate

Probe 2- 3’ Flourescein

Hybridisation- Fluor excited and

transfers energy to

Red Acceptor probe

Red Fluorescence will be detected

42

Real-time PCR assay-3

A Molecular beacon has a Donor

And an acceptor in a stem loop

(Closed) conformation

These are good for detection of small sequence changes/single mutations

As they only hybridise when there is a perfect match at the Target

Hybridisation will result in separation between FLOUR and QUENCHER

FLOURescence will now be emitted and detected by machine

43

Use of FLOURESCENT PROBES IN REAL TIME PCR

MAKES reaction SPECIFIC!44

Types of PCR

• Qualitative PCR

– Allows determination of presence or absence

of target gene

• Quantative PCR

– Allows determination of specific quantity of

target as compared with quantification

standards, e.g. viral load determination

45

Types of PCR

• Quantitative

For viral load determination, HCV, HSV, HIV,

HDV, HBV

Treatment response – therapy and progress

For extent of mutation – such as Bcr/Abl

mutation in Chronic myeloid leukemia

46

15SERIES OF 10-FOLD DILUTIONS

threshold

47

Advantages

•sensitive

•quick

•provides a high copy of genes

•a small sequence can be targeted

Disadvantage

false positives

PCR

48

APPLICATIONS of PCR

•DIAGNOSIS, identification of pathogen

•SPECIATION, e.g. M.avium/ M. intracellulare

•DETECTION OF DRUG RESISTANCE

•IDENTIFICATION OF TOXINS, e.g. Clostridium

•EPIDEMIOLOGICAL STUDIES

genotyping, ribotyping

49

Advantages and Disadvantages of

Realtime PCR assays

• Advantage

– Highly specific and sensitive

– Multiple probe labels (530/640 /705nm) allow

multiplexing of signals and detection of target

and control DNA

– Disadvantage – relatively expensive and

specialised equipment required

50

Summary of this class

• To relate the question (target genetic

material) with the assay required

• To understand how polymerase chain

reaction works

• To understand the methods for visualising

PCR products

• To understand conventional and real time

PCR

• To differentiate qualitative and quantitative

PCR 51