You are employed in a gene therapy laboratory to test the effectiveness of a vector for correcting the DF508 –CFTR mutation. The vector uses CRISPR –CAS9 genome editing to correct the mutated gene in situ (CFTR-CRISPR). An Ussing chamber experiment was performed using nasal airway cells obtained from a patient with the DF508 CFTR mutation which were transformed with either a control vector consisting of an inert CRISPR-CAS-9 construct (CONTROL) or the active, gene correcting CRISPR CAS-9 construct (CFTR-CRISPR). The following transepithelial voltage values were obtained for the drug regimen shown under open circuit and current clamp (injected current of 10mA) conditions: CONTROL Baseline Amiloride Forskolin Inh172 Vte (mV) -78 -2 -4 -3 DVRt (mV) 8 16 7.5 13 R (W.cm-2) 800 1600 750 1300 I (mA.cm-2) 98 1 5 2 CFTR-CRISPR Vte (mV) -38 -6 -23 -9 DVRt (mV) 9 27 6 11 R (W.cm-2) 900 2700 600 1100 I (mA.cm-2) 42 2 38 8 Calculate the electron flow through CFTR for both “CONTROL” and “CFTR-CRISPR”. Note that 1 Ampere = 1 coulomb.second-1 = 6.24×1018second-1 Give your answer as electrons.cm-2.sec-1 to two decimal places followed by power of 10 magnitude (eg 5.66 x1015would give answers of 5.66 and 15)
Molecular Techniques
Molecular techniques are methods employed in molecular biology, genetics, biochemistry, and biophysics to manipulate and analyze nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)), protein, and lipids. Techniques in molecular biology are employed to investigate the molecular basis for biological activity. These techniques are used to analyze cellular properties, structures, and chemical reactions, with a focus on how certain molecules regulate cellular reactions and growth.
DNA Fingerprinting and Gel Electrophoresis
The genetic makeup of living organisms is shown by a technique known as DNA fingerprinting. The difference is the satellite region of DNA is shown by this process. Alex Jeffreys has invented the process of DNA fingerprinting in 1985. Any biological samples such as blood, hair, saliva, semen can be used for DNA fingerprinting. DNA fingerprinting is also known as DNA profiling or molecular fingerprinting.
Molecular Markers
A known DNA sequence or gene sequence is present on a chromosome, and it is associated with a specific trait or character. It is mainly used as a genetic marker of the molecular marker. The first genetic map was done in a fruit fly, using genes as the first marker. In two categories, molecular markers are classified, classical marker and a DNA marker. A molecular marker is also known as a genetic marker.
DNA Sequencing
The most important feature of DNA (deoxyribonucleic acid) molecules are nucleotide sequences and the identification of genes and their activities. This the reason why scientists have been working to determine the sequences of pieces of DNA covered under the genomic field. The primary objective of the Human Genome Project was to determine the nucleotide sequence of the entire human nuclear genome. DNA sequencing selectively eliminates the introns leading to only exome sequencing that allows proteins coding.
You are employed in a gene therapy laboratory to test the effectiveness of a vector for correcting the DF508 –CFTR mutation. The vector uses CRISPR –CAS9 genome editing to correct the mutated gene in situ (CFTR-CRISPR). An Ussing chamber experiment was performed using nasal airway cells obtained from a patient with the DF508 CFTR mutation which were transformed with either a control vector consisting of an inert CRISPR-CAS-9 construct (CONTROL) or the active, gene correcting CRISPR CAS-9 construct (CFTR-CRISPR). The following transepithelial voltage values were obtained for the drug regimen shown under open circuit and current clamp (injected current of 10mA) conditions:
|
CONTROL |
|
Baseline |
Amiloride |
Forskolin |
Inh172 |
|
Vte (mV) |
-78 |
-2 |
-4 |
-3 |
|
|
DVRt (mV) |
8 |
16 |
7.5 |
13 |
|
|
R (W.cm-2) |
800 |
1600 |
750 |
1300 |
|
|
I (mA.cm-2) |
98 |
1 |
5 |
2 |
|
|
CFTR-CRISPR |
Vte (mV) |
-38 |
-6 |
-23 |
-9 |
|
DVRt (mV) |
9 |
27 |
6 |
11 |
|
|
R (W.cm-2) |
900 |
2700 |
600 |
1100 |
|
|
I (mA.cm-2) |
42 |
2 |
38 |
8 |
- Calculate the electron flow through CFTR for both “CONTROL” and “CFTR-CRISPR”. Note that 1 Ampere = 1 coulomb.second-1 = 6.24×1018second-1 Give your answer as electrons.cm-2.sec-1 to two decimal places followed by power of 10 magnitude (eg 5.66 x1015would give answers of 5.66 and 15)
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