Molecular techniques scheme

Molecular techniques

Samadhi Galván | Galia Pérez

Polymerase Chain Reaction (PCR)

DEFINITION:

TYPE OF SAMPLE:

Is technique used to amplify or copy small segments of DNA, in vitro by using instruments designed to manipulate changes in temperature called thermal cyclers.

• Nasopharyngeal (NP) swabs
• Oropharyngeal (OP) swabs

• Sputum

STEPS:

1. Denaturing (15-30 seconds):

2. Annealing (10-30 seconds):

a)Template DNA + all the other core ingredients is heated to 94-95*C.

b)High temperature = break of hydrogen bonds between the bases in the two strands of template = two single strands of DNA (template).

c)Mantain high temperature.

Reaction is cooled to 50-65*C to enable the primers to attach to a specific location on the single-stranded template DNA by way of hydrogen bonding.

3. Extending

(1min = copy 1000 DNA bases):

The heat is increased to 72*C to enable the new DNA to be made by a special Taq DNA polymerase enzyme which adds DNA bases.

72⁰C is the optimum temperature for the Taq polymerase to build the complementary strand.

FINAL:

• These three stages are repeated 20-40 times, doubling the number of DNA copies each time.

• After PCR has been completed, a method called electrophoresis can be used to check the quantity and size of the DNA fragments produced.

REFERENCES:

What is it used for in Molecular Nutrition?

How are the results displayed and interpreted

Differential display polymerase chain reaction has emerged as an instrument of unlimited potential for assessing the manner by which nutrients regulate cell functions.

Determine the right type of food for an individual based on his or her genomic compatibility and therefore aid in avoiding foods that are an inappropriate match and could potentially impact negatively on the individual’s health.

Ct (threshold cycle)

The threshold cycle (Ct) is the cycle number at which the fluorescent signal of the reaction crosses the threshold.

The Ct is used to calculate the initial DNA copy number, because the Ct value is inversely related to the starting

amount of target.

Standard curve

Correlation coefficient (R2)

A dilution series of known template concentrations can be

used to establish a standard curve for determining the initial starting amount of the target template in experimental

samples or for assessing the reaction efficiency.

The correlation coefficient is a measure of how well the data fit the standard curve. The R2

value reflects the linearity of the standard curve.

The threshold of the real-time PCR reaction is the level of signal that reflects a statistically significant increase

over the calculated baseline signal.

Y-intercept

The y-intercept corresponds to the theoretical limit of

detection of the reaction, or the Ct value expected if the

lowest copy number of target molecules denoted on the

x-axis gave rise to statistically significant amplification.

Relative quantification

Exponential phase

Relative quantification describes a real-time PCR experiment in which the expression of a gene of interest in one sample (i.e., treated) is compared to expression of the same gene in another sample

It is important to quantify your real-time PCR reaction in the early part of the exponential phase as opposed to in the later cycles or when the reaction reaches the plateau.

Melting curve (dissociation curve

Slope

The slope of the log-linear phase of the amplification reaction is a measure of reaction efficiency.

A melting curve charts the change in fluorescence observed when double-stranded DNA (dsDNA) with incorporated dye molecules dissociates, or “melts” into single-stranded DNA

(ssDNA) as the temperature of the reaction is raised.

Efficiency

Bass, JJ. et al (2017). An overview of technical considerations for Western blotting applications to physiological research. Recovered from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5138151/

Lifetech. (2012). Real-time PCR handbook. Recovered from https://www.gene-quantification.de/real-time-pcr-handbook-life-technologies-update-flr.pdf

ELISA blood test. (n.d.). Medlineplus. Medline puls.. Retrieved from https://medlineplus.gov/ency/article/003332.htm#:~:text=ELISA%20stands%20for%20enzyme%2Dlinked,detects%20harmful%20substances%2C%20called%20antigens

Kinman, T. (2018). ELISA. Helathline. REtrieved from https://www.healthline.com/health/elisa#preparation

ELISA results. (n.d.). Mybiosource. My biosource. Retreived from https://www.mybiosource.com/elisa-results

Western blot. (n.d.). Nuture. Nature. Retrieved from https://www.nature.com/scitable/definition/western-blot-288/

Mahmood, T., & Yang, P. C. (2012). Western blot: technique, theory, and trouble shooting. North American journal of medical sciences, 4(9), 429–434. https://doi.org/10.4103/1947-2714.100998

A PCR efficiency of 100% corresponds to a slope of -3.32, as determined by the following equation: Efficiency = 10(-1/slope) -1

Dynamic range

This is the range over which an increase in starting material concentration results in a corresponding increase in amplification product.

Absolute quantification

Absolute quantification describes a real-time PCR experiment in which samples of known quantity are serially diluted and then amplified to generate a standard curve.

WESTERN BLOT

Laboratory method used to detect specific protein molecules from among a mixture of proteins

STEPS

Principle: Cell lysis to extract protein.

• Prepare sample (saliva or tissue) with detergent to unfold proteins and coat them (-)
• Gel electrophoresis: separate cells by sized
• Transfer from gel to blotting membrane
• Membrane carries protein bands on the gel
• Blocking: Prevents non-specific reactions
• Membrane incubated with the primary antibody (binds to protein)
• Secondary antibody binds to reporter enzyme and produces color

How are the results displayed and interpreted

What is it used for in Molecular Nutrition?

1. Quantify against your control.

2.Use ImageJ to see if there's an increased intensity in the bands (increased expression).

3. Analize if antibodies shows bands at different molecular weights as a non-specific binding, otherwise analize if they're breakdown products.

Application to skeletal muscle and exercise physiology. Understanding of molecular pathways involved in the regulation of transcription and translation by exercise and nutrition in health, ageing and disease.

ELISA

Enzyme-Linked Immunoassay

DEFINITION

RESULTS

Quantitative: Comparison to standard curve.

Qualitative: yes or no presence of antigen.

Semi quantitative: signal intensity dependent on amount

Test used to detect antibodies in the blood.

Can diagnose:

• HIV
• Lyme disease
• Pernicious anemia
• Etc.

TYPES

• Indirect ELISA
• Direct ELISA
• Sandwich ELISA
• Competitive ELISA

USE IN MOLECULAR NUTRITION

STEPS

Can be used to detect food allergens

• Extraction of blood sample.
• Immobilization of antigen on solid surface.
• Antigen is complex-ed to a detection antibody conjugated with a molecule for detection.