polymerase chain reaction pcr l.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Polymerase Chain Reaction (PCR) PowerPoint Presentation
Download Presentation
Polymerase Chain Reaction (PCR)

Loading in 2 Seconds...

play fullscreen
1 / 72

Polymerase Chain Reaction (PCR) - PowerPoint PPT Presentation


  • 456 Views
  • Uploaded on

Polymerase Chain Reaction (PCR) Outline 1. DNA 2. PCR Targets Denaturing Primers Annealing Cycles Requirements Outline 3. Applications of PCR Neisseria gonorrhoeae Chlamydia HIV-1 Factor V Leiden Forensic testing 4. Extraction of DNA for Factor V Outline

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Polymerase Chain Reaction (PCR)' - jana


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
outline
Outline

1. DNA

2. PCR

  • Targets
  • Denaturing
  • Primers
  • Annealing
  • Cycles
  • Requirements
outline3
Outline

3. Applications of PCR

  • Neisseria gonorrhoeae
  • Chlamydia
  • HIV-1
  • Factor V Leiden
  • Forensic testing

4.Extraction of DNA for Factor V

outline4
Outline

5. DNA Detection for Factor V

6. PCR Results for Factor V

7. Conclusion

slide5
DNA

DNA is a nucleic acid that is composed of two complementary nucleotide building block chains.

The nucleotides are made up of a phosphate group, a five carbon sugar, and a nitrogen base.

slide6
DNA
  • DNA Sugar
    • Deoxyribonucleic acid
  • RNA Sugar
    • Ribonucleic acid
slide7
DNA

DNA has four nitrogen bases.

  • Two are purines ( 2 ringed base )
    • Adenine ( A ), Guanine ( G )
  • Two are pyrimidines ( 1 ringed base )
    • Cytosine ( C ), Thymine ( T )
slide8
DNA

These four bases are linked in a repeated pattern by hydrogen bonding between the nitrogen bases.

The linking of the two complementary strands is called hybridization.

slide9
DNA

A purine always links with a pyrimidine base to maintain the structure of DNA.

Adenine ( A ) binds to Thymine ( T ), with two hydrogen bonds between them.

Guanine ( G ) binds to Cytosine ( C ), with three hydrogen bonds between them.

slide10
DNA

Example of bonding pattern.

  • Primary strand

CCGAATGGGATGC

GGCTTACCCTACG

  • Complementary strand
dna molecule
DNA Molecule

Adenine

Thymine

Guanine

Cytosine

slide12
PCR

PCR is a technique that takes a specific

sequence of DNA of small amounts and

amplifies it to be used for further testing.

pcr targets
PCR 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.

pcr targets14
PCR Targets

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.

pcr denaturing
PCR Denaturing

Denaturing is the first step in PCR, in which

the DNA strands are separated by heating to

95°C.

pcr primers
PCR Primers

Primers range from 15 to 30 nucleotides, are

single-stranded, and are used for the

complementary building blocks of the target

sequence.

pcr primers17
PCR Primers

A primer for each target sequence on the end

of your DNA is needed. This allows both

strands to be copied simultaneously in both

directions.

pcr primers18
PCR Primers

TTAACGGCCTTAA . . . TTTAAACCGGTT

AATTGCCGGAATT . . . . . . . . . .>

and

<. . . . . . . . . . AAATTTGGCCAA

TTAACGGCCTTAA . . . TTTAAACCGGTT

pcr primers19
PCR Primers

The primers are added in excess so they will

bind to the target DNA instead of the two

strands binding back to each other.

pcr annealing
PCR Annealing

Annealing is the process of allowing two

sequences of DNA to form hydrogen bonds.

The annealing of the target sequences and

primers is done by cooling the DNA to 55°C.

pcr taq dna polymerase
PCR Taq DNA Polymerase

Taq stands for Thermus aquaticus, which is a

microbe found in 176°F hot springs in Yellow

Stone National Forest.

pcr taq dna polymerase22
PCR Taq DNA Polymerase

Taq produces an enzyme called DNA

polymerase, that amplifies the DNA from the

primers by the polymerase chain reaction, in

the presence of Mg.

pcr cycles review
PCR Cycles Review
  • Denaturalization: 94°- 95°C
  • Primer Annealing: 55°- 65°C
  • Extension of DNA: 72°
  • Number of Cycles: 25-40
pcr requirements
PCR Requirements
  • Magnesium chloride: .5-2.5mM
  • Buffer: pH 8.3-8.8
  • dNTPs: 20-200µM
  • Primers: 0.1-0.5µM
  • DNA Polymerase: 1-2.5 units
  • Target DNA:  1 µg
applications of pcr
Applications of PCR
  • Neisseria gonorrhea
  • Chlamydia trachomatis
  • HIV-1
  • Factor V Leiden
  • Forensic testing and many others
applications of pcr31
Applications of PCR

Neisseria gonorrhea and Chlamydia trachomatis are two of the most common sexually transmitted diseases. The infections are asymptomatic and can lead to pelvic inflammatory disease, salpingitis in women, epididymitis in men, infertility, and ectopic pregnancy.

applications of pcr32
Applications of PCR

Specimens include endocervical swabs,urethral swabs, and urine samples.

The swabs are placed in a vial with transport buffer containing  50mM MgCL2 and sodium azide as a preservative.

applications of pcr33
Applications of PCR

The swab specimens can be stored 2-30°C for 4 days or frozen at -20°C.

The urine samples are refrigerated at 2-8°C or stored at -20°C.

A target sequence is chosen for both, amplified with polymerase, and then evaluated with an enzyme immunoassay.

applications of pcr34
Applications of PCR

HIV-1 and Factor V Leiden also have a specific target sequence amplified, and then quantitated by using a microwell probe, horse-radish peroxidase enzyme, and chromogen substrate.

applications of pcr35
Applications of PCR

The HIV-1 test is used as a monitor of the severity of the virus. The HIV-1 causes a depletion of CD4+ T lymphocytes, causing immunodeficiency, multiple opportunistic infections, malignancies, and death.

applications of pcr36
Applications of PCR

The HIV-1 specimen is plasma collected in EDTA that must be separated from the cells within 6 hours.

Heparin cannot be used as an anticoagulant because it inhibits PCR.

applications of pcr37
Applications of PCR

A 142 base target sequence in the HIV-1 gag gene is converted from RNA to complementary DNA, and to double stranded DNA using Thermus thermophilus DNA polymerase in the presence of manganese and buffers, which performs the reverse transcription and the amplification steps simultaneously.

applications of pcr38
Applications of PCR

The standard specimen procedure can quantitate HIV-1 RNA in a range of 400-75,000 copies/mL.

applications of pcr39
Applications of PCR

Factor V Leiden is the Factor V in the coagulation cascade.

Factor V is a genetic point mutation that causes increased risk of life-threatening blood clots.

The mutation causes the Factor V molecule to be unresponsive to the natural anti-coagulant protein C.

applications of pcr40
Applications of PCR

Factor V Leiden shifts the patient’s hemostatic balance to thrombosis.

Factor V mutation gives an increase risk of venous thrombosis in a homozygous person, during pregnancy, surgery, or while using oral contraceptives.

applications of pcr41
Applications of PCR

Thrombosis - is the development of a blood clot that occurs in 20-40% of patients with venous thrombosis.

Thrombophilia - a tendency towards clotting that occurs in 40-65% of adults with unexplained thrombophilia.

Protein C - a naturally occurring anti-

coagulant that occurs in 95-100% of people with activated protein C resistance.

application of pcr
Application of PCR

Treatment for patients with Factor V Leiden mutations are to give lifelong coumadin.

Women with the mutation should not take oral contraceptives, and they have increased risk of thrombosis during pregnancy.

applications of pcr43
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.

applications of pcr44
Applications of PCR

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.

applications of pcr46
Applications of PCR

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.

extraction of dna for factor v
Extraction of DNA for Factor V

The anticoagulant tube with the patient’s blood sample should be centrifuged to separate it into the layers of plasma, Buffy coat, and the RBCs.

The buffy coat is used for the extraction because it contains WBCs, which are nucleated and possess the DNA.

extraction of dna for factor v49
Extraction of DNA for Factor V

Extract and discard plasma, taking care not to remove

the buffy coat.

extraction of dna for factor v50
Extraction of DNA for Factor V

Carefully extract 200µl of buffy coat from each sample

and place in designated tube.

extraction of dna for factor v51
Extraction of DNA for Factor V

Add 25µl of

protease

to each tube.

Add 200µl of

lysis buffer

to each tube.

extraction of dna for factor v52
Extraction of DNA for Factor V

Vortex each tube

for 15 sec. to

ensure proper

mixing.

extraction of dna for factor v53
Extraction of DNA for Factor V

Incubate each tube

for 10 min. at 56°C.

extraction of dna for factor v54
Extraction of DNA for Factor V

Centrifuge each to

remove any mixture

that may be on the lid.

Add 210µl of ethanol,

vortex and then

centrifuge again.

extraction of dna for factor v55
Extraction of DNA for Factor V

Add sample mixture to column tube and centrifuge

for 1 min.

extraction of dna for factor v56
Extraction of DNA for Factor V

Column Tube

This column section fits into

another tube, which catches

the eluted substances.

This is the

column.

This is the eluted

sample that is

discarded.

extraction of dna for factor v57
Extraction of DNA for Factor V

The column portion is inserted into a new tube and

washed twice, each time 500µl of buffer is used to elute

substances

adsorbed to

the column

that are not

DNA.

extraction of dna for factor v58
Extraction of DNA for Factor V

The column portion is then centrifuged for 1 minute to remove excess washing buffer.

Next, 100L of eluting buffer is added.

  • This is incubated for 5 minutes at 25°C, and then centrifuged.
  • The elute is kept this time because it contains the DNA.
extraction of dna for factor v59
Extraction of DNA for Factor V
  • During the 5 minute incubation, the master mix should be prepared.
  • Master Mix
  • 10x Buffer - 10 µl
  • MgCl - 6 µl
  • dNTP mix - 0.8 µl
  • of each nucleotide
  • F5F primer - 2 µl
  • F5R primer - 2 µl
  • Taq polymerase - 0.5 µl
  • Sterile HO - 73.7 µl
extraction of dna for factor v60
Extraction of DNA for Factor V

Place 5µl of patient sample

and 95µl of master mix in

vials and place these vials

in a PCR panel, which

will then be placed in

the thermocycler for the

DNA amplification cycles.

dna detection for factor v
DNA Detection for Factor V

To prove that the DNA was amplified a DNA enzyme immunoassay (DEIA) is performed.

The test is done by denaturing the amplified DNA and adding it to probe-coated microtiter wells.

If the amplified DNA sequences are complementary to the probes, double stranded hybrids will form.

dna detection for factor v62
DNA Detection for Factor V

A mouse monoclonal antibody is added that will only bind to double-stranded DNA hybrids.

Positive and negative wells are detected colorimetric by adding an enzyme (conjugated protein A with horseradish peroxidase), substrate, and chromogen.

The is incubated at room temp. away from light for 30 mins. to develop the color.

dna detection for factor v63
DNA Detection for Factor V

Upon finishing the

incubation, the

panel looks like

this.

dna detection for factor v64
DNA Detection for Factor V

The color is then

stopped, by the

addition of

200µl of an

acidic stop

solution.

dna detection for factor v65
DNA Detection for Factor V

The plate is then placed in the automated reader,

where each well is read spectrophotometrically.

dna detection for factor v66
DNA Detection for Factor V

Each well is read at 450nm and then at 630nm.

The difference between the two absorbance:

A at 450nm

- A at 630nm

Final A value

A positive hybridization result is indicated by an

absorbance value greater that the mean

negative control plus 0.150 absorbance units.

dna detection for factor v67
DNA Detection for Factor V

The machine

then gives

you a read out,

from which

you calculate

the patient

results.

dna detection for factor v68
DNA Detection for Factor V

Obtain a ratio

from the values:

0.982

0.041

which will give

you

23.95

Homozygous

Normal = >5.0

conclusion
Conclusion

PCR is not only vital in the clinical laboratory by amplifying small amounts of DNA for STD detection, but it is also important for genetic predisposing for defects such as Factor V Leiden.

The PCR technology can also be employed in law enforcement, genetic testing of animal stocks and vegetable hybrids, and drug screening along with many more areas.

references
References
  • Assay: Abbott Laboratories

Neisseria gonorrhoeae

List # 8A48-81

  • Assay: Roche Diagnostics

Amplicor HIV-1 Monitor Test

List# 83088

  • Assay: GEN-ETI-K (DiaSorin)

DEIA-Factor V Leiden

Catalog# PS5096

references71
References
  • Alberts, Brown,Johnson, Lewis, Raff, Roberts, Walter. Use of PCR in Forensic Science. 1998. Online. Internet. 18 Jan. 2001. Available http://www.accessexcellence.org/AB/GG/

forensci_PCR.htm.l

  • Brown, John C. What The Heck Is PCR? 1995. Online. Internet. 18 Jan. 2001. Available

http://falcon.cc.ukans.edu/~jbrown/pcr.html

  • Photographs: Courtesy of UMC clinical lab and Tom Wiggers.
references72
References
  • Ronald H. Holton, Ph.D.:
    • Molecular Diagnostics in the Clinical Laboratory
    • Molecular Biology in the Clinical Laboratory
    • Molecular Pathology: Basic Methodologies and Clinical Applications
    • Expanding applications of PCR, by Peter Gwynne and Guy Page