2. RESPIRATION
(1) Pulmonary ventilation
(2) External respiration
Respiratory gas transport
(3)
Internal respiration
(4)
Breathing
: means
movement
of air In and
Outof thebody
Oxygen loading and
carbondioxide
loading
Transportation of gasesby blood stream
Exchange between capillary and body ce
3. (1) BREATHING (PULMONARY VENTILATION)
Inspiration: Diaphragm flattens creates a vacuum pulling
air into the lungs
Expiration: Muscles relax and push air out of the lungs
7. Pulmonary Function Tests
Definition:
Pulmonary function tests (PFTs) are noninvasive tests that show how well the
lungs are working. The tests measure lung volume, capacity, rates of flow, and
gas exchange. This information can help your healthcare provider diagnose
and decide the treatment of certain lung disorders
Types of PFTs
1-Spirometry measures the rate of air flow and estimates lung size. For
this test, you will breathe multiple times, with regular and maximal effort,
through a tube that is connected to a computer. Some people feel lightheaded
or tired from the required breathing effort.
.
8. 2-Lung volume tests are the most accurate
way to measure how much air your lungs can
hold.
3-Lung diffusion capacity assesses how well
oxygen gets into the blood from the air you
breathe.
3-Pulse oximetry estimates oxygen levels in
your blood
4-Arterial blood gas tests
5-Fractional exhaled nitric oxide tests
11. •Arterial blood gas tests
•Arterial blood gas tests directly measure the levels of gases, such as oxygen and
carbon dioxide, in blood.
Collection and handling of arterial blood gases:
1)The specimen for blood gases and pH should be arterial or arterialized
capillary blood
2)All air bubbles should be removed.
3)Air contamination will reduce the CO2 and increases the O2 in the sample
due to the difference in the PO2 and PCO2 tension of these gases in the
atmosphere.
4)Use the correct amount of heparin (0.05 mg heparin/ml blood).
5)The specimen must be placed in ice water until analysis or examined
immediately.
12. Importance of pulmonary function tests?
•They are important for detection of :
•Allergies
•Respiratory infections
•Trouble breathing from injury to the chest or a recent surgery
•Chronic lung conditions, such as asthma, bronchiectasis,
emphysema, or chronic bronchitis
•Asbestosis, a lung disease caused by inhaling asbestos fibers
•Restrictive airway problems from scoliosis, tumors, or
inflammation or scarring of the lungs
•Sarcoidosis, a disease that causes lumps of inflammatory cells
around organs, such as the liver, lungs, and spleen
•Scleroderma, a disease that causes thickening and hardening of
connective tissue
13. •the 5' to 3' exonuclease activity of
the Taq polymerase degrades the probe that has
annealed to the template.
• Degradation of the probe releases the
fluorophore from it and breaks the proximity to
the quencher, thus relieving the quenching effect
and allowing fluorescence of the fluorophore.
•Hence, fluorescence detected in the quantitative
PCR thermal cycler is directly proportional to the
fluorophore released and the amount of DNA
template present in the PCR.
14. 1). In this plot, the number of
PCR cycles is shown on the
x-axis, and the fluorescence
from the amplification
reaction, which is
proportional to the amount of
amplified product in the tube,
is shown on the y-axis.
15. Initially, fluorescence remains at background levels, and increases in
fluorescence are not detectable (cycles 1–18, Figure 1) even though product
accumulates exponentially.
Eventually, enough amplified product accumulates to yield a detectable
fluorescence signal.
The cycle number at which this occurs is called the quantification cycleCq, or.
Threshold cycle Ct
, The Threshold Cycle (Ct) or the quantification cycle (Cq): is the number
of cycles required for the fluorescent signal to cross the threshold (exceeds
background level), or it is the PCR cycle at which the amplification curve
intercepts with the threshold line
16. The Cq of a reaction is determined mainly by the amount of template present at
the start of the amplification reaction.
If a large amount of template is present at the start of the reaction, relatively
few amplification cycles will be required to accumulate enough product to give a
fluorescence signal above background. Thus, the reaction will have a low, or
early, Cq.
In contrast, if a small amount of template is present at the start of the reaction,
more amplification cycles will be required for the fluorescence signal to rise
above background. Thus, the reaction will have a high, or late, Cq.
This relationship forms the basis for the quantitative aspect of real-time PCR.
17. Blue sample Ct value=23. Green sample Ct value=28. Therefore the blue
sample contained 32 (25 ) times more of the gene of interest than the green
sample.
18. comparison between real time PCR and traditional PCR
•Traditional PCR: uses agarose gel
for detection of PCR amplification at
the plateau phase or end-point of the
PCR reaction.
•If measurements were taken at the
plateau phase, the data would not truly
represent the initial amounts of starting
target material.
•Real-Time PCR provides fast, precise
and accurate results. It is designed to
collect data as the reaction is
proceeding (exponential phase ),
which is more accurate for DNA and
RNA quantitation and does not require
post PCR methods
19. 2-Reverse Transcription Quantitative PCR (RT-qPCR)
• The starting template material is RNA not DNA ( as in PCR assays for the
diagnosis of viral infections)
• RNA cannot serve as a template for PCR, (RNA is not a substrate for the
Taq DNA polymerases commonly utilised in PCR.) Therefore reverse
transcription is combined with PCR to convert RNA into a complementary
DNA (cDNA)) suitable for PCR
• The first step in this procedure is to convert the RNA molecules into single-
stranded complementary DNA (cDNA) (Figure 9.20). Once this preliminary
step has been carried out, the PCR primers and Taq polymerase are
added and the experiment proceeds exactly as in the standard technique
20.
21. The RT reaction
• one-step procedure: The reverse transcription process and the PCR
can be performed at the same time where all the components for RT and
PCR are present from the start, i.e., RNA, primers, dNTP’s, PCR buffer,
RT and Taq enzymes (or an enzyme that can perform both functions).
Two steps procedure: involves creating cDNA first by means of a
separate reverse transcription reaction and then adding the cDNA to the
PCR reaction (more preferred better control the assay and to perform a
reaction titration)
22. DNA sequencing:
• Def: DNAsequencingis the processof determining the sequence of nucleotide
bases (As,T
s,Cs,and Gs)in apiece of DNA
•There are several procedures for DNA sequencing:
1. Maxam and Gilbert Method: method based on chemical
modification of DNA and subsequent cleavage at specific bases
2. ChainTermination(Sanger)Sequencing:A modified DNA replication
reaction. Growing chains are terminated by dideoxynucleotides
3. Pyro sequencing : new generation method that detects light
emitted during the sequential addition of nucleotides during the
synthesis of a complementary strand of DNA.
23. Sanger sequencing: the most popular method first devised by Fred
Sanger and colleagues in the mid-1970
•DNA to be sequenced serves as a template for DNA synthesis.
•A DNA primer is designed to be a starting point for DNA synthesis by
DNA polymerase on the strand of DNA to be sequenced.
•Four individual DNA synthesis reactions are performed.
•The four reactions include normal A, G, C, and T deoxynucleotide
triphosphates (dNTPs), and each contains a low level of one of four
dideoxynucleotide triphosphates (ddNTPs): ddATP, ddGTP, ddCTP, or
ddTTP.
24. •The four reactions can be named A, G, C
and T, according to which of the four
ddNTPs was included.
•When a ddNTP is incorporated into a
chain of nucleotides, synthesis terminates.
This is because the ddNTP molecule lacks
a 3' hydroxyl group, which is required to
form a link with the next nucleotide in the
chain.
•Since the ddNTPs are randomly
incorporated, synthesis terminates at many
different positions for each reaction
25. •Following synthesis, the products of the A, G, C, and T reactions are
individually loaded into four lanes of a single gel and separated using gel
electrophoresis
•The bands of the gel are detected, and then the sequence is read from
the bottom of the gel to the top, including bands in all four lanes.
For instance, if the lowest band
across all four lanes appears in the A
reaction lane, then the first
nucleotide in the sequence is A.
Then if the next band from bottom to
top appears in the T lane, the
second nucleotide in the sequence
is T, and so on.
