Test - 1 Flashcards
What is used to amplify DNA fragment?
PCR
What is used to amplify DNA?
Thermostable Taq DNA polymerase enzyme
What are the PCR components?
DNA sample, Primers, Nucleotides, Taq polymerase, Mix Buffer, PCR Tube
What are the steps of the PCR process?
Denaturing(95C), Annealing(55C) and Extension(72C)
Process to seperate DNA
Agarose Gel Electrophoeresis is used to seperate DNA based on molecular weight
What is the 3D matrix used in DNA seperation?
Agarose
what electrode does DNA move to during electrophoresis?
Negative to positive
What is the stain used during separation of DNA and what light is used to observe it?
Ethidium Bromide and Ultraviolet Light
Method to separate proteins based on molecular weight
Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Charge imparted during SDS-PAGE process on proteins
Negative
Gel used SDS-PAGE is made of
Gel is made of Polyacrylamide and protein mixture is loaded on top
Energy in 1gm of carbs, proteins and fats?
4, 4, 9 calories
Five carbon aldoses in order
Ribose, Arabinose, Xylose and Lyxose
Six carbon aldoses in order
Allose, Altose, Glucose, Mannose, Gulose, Idose, Galactose and Talose
Five carbon Ketoses in order
Ribulose and Xylulose
Six carbon ketoses in order
Psicose, Fructose, Sorbose and Tagatose
Monosaccharides in maltose
alpha glucose and alpha glucose
Monosaccharides in Lactose
beta Galactose(1) and alpha Glucose(4)
Monosaccharides in Sucrose
beta fructose(2) to alpha glucose(1)
Monosaccharides in Trehalose
alpha Glucose(1) to Alpha Glucose(1)
The branching in amylopectin is of the type
(alpha1 to alpha6)
Role of Dextran
extracellular adhesive in baceteria
Role of Peptidoglycan
gives rigidity and strength to cell envelope in bacteria
Significance of Hyaluronan
Extracellular matrix of skin and connective tissue, viscosity and lubrication of joints in vertebrates
What are phospho-sphingolipids made of?
Sphingoline, Fatty acid and (PO4 - Choline)
What are GlycoSphingolipids made of?
Sphingoline, Fatty acid and Mono- or Oligo- saccharide
What are Galactolipids?
Galactolipids are Glycolipids made of Glycerol, 2 Fatty Acids and a (Mono- or Oligo- Saccharide - SO4)
What are Archeal ether lipids made of?
(Glycerol - PO4) connected to another glycerol by two Diphytanyl ether linkages which itself is connected to another Glycerol by a PO4
What are simple triacylglycerols?
Glycerols with the same fatty acids at all 3 positions
The three simple tryacylglycerols 16:0, 18:0 and 18:1 are
tripalmitin, tristearin and triolein
What is fat tissue?
Adipocytes
What is a primer?
Primer is a short nucleic acid sequence that provides a starting point for DNA synthesis
What are omega three fatty acids?
Poly Unsaturated Fatty Acids with a double bond between C-3 and C-4 from the methyl side are called omega 3 fatty acids
Why are omega-3 PUFAs important
A dietary ratio of 1:1 to 1:4 of omega-3 and omega-6 should be maintained to avoid cardiovascular disease. ratio in diet in average north american is 1:10 to 1:30
At room temperature saturated fatty acids 12:0 to 24:0 and unsaturated fatty acids are
waxy solids and liquids
What is good cholesterol and bad cholesterol?
LDL is bad🤢 and HDL is good (trans fat increases LDL)
How many types of membrane lipids are there and what are they?
5 and glycerophospholipids, (galactolipids and sulfolipids), tetraether lipids, sphingolipids and sterols
Glycerophospholipids structure
two fatty acids joined to glycerol
galactolipids and sulfolipids structure
two fatty acids esterified to glycerol, but lack the phosphate of phospholipids.
tetraether lipids
two very long alkyl chains are ether-linked to glycerols at both ends
sphingolipids structure
a single fatty acid is joined to fatty amine, sphingosine
sterols
rigid system of four fused hydrocarbon rings
What type of lipid determines blood group?
Glycosphingolipid
What is the oligosaccharide in the lipid that determines the bloodgroup that gives O,A.B antigen?
O Antigen (Glu - Gal - GlcNAc - Gal) - Fuc A Antigen (Glu -Gal - GlcNAc - Gal)( - Fuc)( - GalNAc) B Antigen (Glu -Gal - GlcNAc - Gal)( - Fuc)( - Gal)
What is the primary structure of a protein?
the linear sequence of amino acids: Gly-Ala-Lys-Trp
What is the secondary order of proteins?
stable arrangement of amino acids giving rise to recurring structural patterns, such as α-helix and β-sheet.
What secondary structure is present in myoglobin?
alpha helix
Tertiary structure of proteins
three-dimensional folding of a polypeptide chain.ular.
Quaternary structure of proteins
when a protein has two or more polypeptide chains, their arrangement in space.
Groups other than amino acids are called
Prosthetic Groups
protein with carb prosthetic group
Immunoglobin G
protein with phosphate prosthetic group
Casein of Milk
protein with flavo nucleotide prosthetic group
Succinate dehydrogenase
protein with zinc prosthetic group is
Alcohol dehydrogenase
protein with calcium pros group
Calmodulin
protein with molybdenum pros group
dinitrogenase
protein with copper pros group
Plastocyanin
components of nucleotide
Nitrogenous Base, Pentose Sugar and one or more phosphates
Nucletide without phosphate group is called
Nucleoside
DNA contains which pentose sugar
2’-deoxy-D-ribose
the pentose sugar in RNA is
D-ribose
Number of hydrogen bonds in adenine-thymine bond
two
number of hydrogen bonds in Guanine-cytosine bond
3
Erwin Chargaff’s Rules are
1) The base composition of DNA generally varies from one species to another.
2) DNA specimens isolated from different tissues of the same species have the same base composition.
3) The base composition of DNA in a given species does not change with an organisms’s age, nutritional status, or changing environment.
4) In all cellular DNA, A=T and G=C. (A+G = T+C)
How far are vertically stacked bases in DNA?
3.4A
How are the strands in DNA aligned?
Anti-Parallel
What are the 3 forms of DNA and which is the most stable form?
A, B and Z. B being the most stable
What does RNA do in DNA synthesis and what types of RNA are involved?
RNA carries genetic information from DNA to the protein biosynthetic machinery of the ribosome.
By the process of transcription, RNA is synthesized from the DNA template.
Three types of RNAs involved in protein synthesis:
mRNA: Messenger RNA
tRNA: Transfer RNA
rRNA: Ribosomal RNA
Tertiary Structure of RNA
Single Strands
Bulge
Internal loop
Hairpin
What are the three types of cloning?
Reproductive cloning/organism cloning (legally restricted)
Therapeutic cloning/stem cell cloning
Molecular cloning/DNA cloning
Removal of nucleus from the egg is called
Enucleation
Circular piece of DNA used in cloning is called?
Plasmid
What is a plasmid?
A plasmid is a small, extrachromosomal DNA within a cell that is physically separated from chromosomal DNA and can replicate independently.
What is the full form of REs?
RE = Restriction Endonucleases
How many types of REs are there and what are they?
- Type 1 REs cleave at random sites
Type 3 REs cleave DNA about 25bp from recognition site
Type 2 REs cleave DNA with recognition site itself. They dont require ATP. They also make staggered or cleaved ends
What are the ways to introduce plasmids into bacterial cells?
Transformation: The cells (often E. coli) and plasmid DNA are incubated together at 0 C in a calcium chloride solution, then subjected to heat shock by rapid shifting the temperature to between 37 C and 43 C.
Electroporation: The cells (often E. coli) are incubated with plasmid DNA are subjected to high-voltage electric pulse. This approach transiently renders the bacterial membrane permeable to large molecules.
What is ori?
Origin of replication (ori), a sequence where replication is initiated by
cellular enzymes
Size of plasmids ranges from?
5000 to 400000 bp
Flourescence has a lower or higher wavelength than absorbed light?
Higher obv🤷♀️
What is the difference between the excitation and emission peaks called?
Stokes shift
Describe protein labelling strategies
1) Genetic encoding: C-terminal or N-terminal
pros: specificity
cons: time taking
2) Antibody based detection
pros: quick
cons: not suitable for live cells
non specific
3)Covalent modification
1) Using Self-labelling enzyme derivatives
2) Using chemical labelling
pros: easy and efficient
cons: suitable for in-vitro studies only
3)Enzymatic labelling:
Fast Efficient and selective for both in-vivo and vitro targetting whole proteins or cells
Where was GFP first discovered?
In the jellyfish Aequorea victoria
What causes the fluorescence of GFP?
Fluorescence is due to amino acids at positions S65. T66 and G67
What is quantum yield?
Ratio of number of photons absorbed to number of photons emitted
Disadvantages of fluorescent proteins over dyes
Use of FPs requires genetic engineering, a time consuming process
Fusion of protein of interest with FP increases its mol. weight by ~27 kD, which may affect protein function.
Fast photobleaching compared to fluorescent dyes
Limited spectral range compared to fluorescent dyes
Less brightness compared to fluorescent dyes
Most FPs tend to oligomerize
What is the first fluorescent dye invented?
fluorescein
Fluorescence recovery after photobleaching (FRAP)
Used to study protein mobility in cells and diffusion dynamics
What is the formula for the diffusion coefficient
D = 0.224\frac{r^2_n}{\tau^\frac{1}{2}}
What is mobile fraction?
Mobile fraction: fraction of fluorescent protein diffusing into bleached region during the time course of the experiment.
What is immobile fraction?
Immobile fraction: fraction of immobile bleached molecules within the bleach spot that don’t allow fluorescent molecules to occupy their space.
Efficiency of FRET based on distance is
inversely proportional to sixth power of dsitance
Efficiency of FRET based on distance is
inversely proportional to sixth power of distance
What is FRET
Forster or Fluorescence Resonance Energy Transfer (FRET) is a mechanism describing energy transfer between two light-sensitive molecules (chromophores).
FRET is used to check protein-protein, protein-DNA interactions and protein conformation dynamics.
What are optical tweezers?
Optical tweezers use tightly focussed light beam (IR laser) to hold and manipulate microscopically small objects.
What are optical tweezers?
Optical tweezers use tightly focussed light beam (IR laser) to hold and manipulate microscopically small objects.
What does DNA ligase do and how?
DNA ligase is used to join two DNA molecules. It forms phosphodiester bonds between the DNA molecules.
A vector digested by an RE can only be ligated to insert digested by same RE
Features of BACs?
They can clone larger DNA fragments
They have stable ori that limits the number of plasmids in a cell
BACs contain a selection and screening marker
What is LacZ and what does it do?
LacZ stands for beta-galactosidase which converts X-gal(white) to a blue product. If LacZ is intact colony will be blue otherwise will be white
Plasmids usable in more than 2 species is called
Shuttle vector
Elements of YAC
yeast Ori, two selectable markers, and specialized sequences (derived from centromeres and telomeres) needed for stability and proper segregation of chromosomes at cell division.
What are expression vectors?
Cloning vectors with the transcription and translation signals needed for the regulated expression of a cloned gene are called expression vectors.
What reduces mobile fraction?
Aggregation of proteins, binding and diffusional barries reduce mobile fraction
Stages of DNA packing
DNA > Nucleosomes > Chromatine > Chromatine Loops > Condensed Chromatine Loops > Chromosome
Phases of mitosis are
Interphase, Prophase,Mrtaphase,Anaphase and Telophase
Is chromosome organization non-territorial or territorial?
Territorial
Steps of laser-UV-microirradiation
1) The nucleus of the live cell was microirradiated in G1
2) Pulse-labeled with 3H thymidine
3) Fixed immediately
4) Autoradiography
What are integral proteins of the nuclear envelope?
Lamina and emerin. They are linked to rare genetic disorders called nuclear envelopathies.
Give examples of envelopathies
Hutchinson-GIlford progeria syndrome and Emery-Dreifuss muscular dystrophy caused by repositioning of chromosomes 13 and 18
Steps of FISH
Fix > Permeablize > Denaturation > Hybridization > Wash to remove unbound and non-specific probes > DAPi > Coverslip
Types of probes for DNA FISh
Centromere probes
Whole Chromosome
Telomere
Locus
What does oxidoreductase enzyme do?
Transfer of electrons
Transferase enzyme
Group Transfer reaction
Hydrolases
Hydrolysis reactions
Lyases
Cleavage of C-C,C-O,C-N
Isomerases enzymes
Yields isomeric forms
Ligases
Formation of C-C,C-S,C-) and C-N by cleavage of ATP
Relation between Substrate Concentration [S] and Reaction Rate[V_0]
V_0 = \frac{V_{max}[S]}{K_m + [S]}
where K_m is the Michealis constant
What is the michealis Menten equation
V_0 = (V_max[S])/(K_m + [S])
What is the Lineweaker Burk Equation
1/V_0 = K_m/(V_max[S]) + 1/V_max
Specificity Constant si
K_cat/K_m
Unit of enzyme activity is
katal = 1micromole/min = 16.67nanomole/s
