Molecular biology Flashcards
Characteristics of DNA to act as vital genetic material
Accurate replication of DNA
Transmission from 1 generation to the other
Ability to store and express hereditary information
Diameter of a DNA double helix
2 nm
Length of 1 complete turn
3.4 nm
Number of base pairs present in 1 complete turn
10 base pairs
Double helical structure was found by
James Watson and Francis Crick based on X-ray crystallography by Rosalind Franklin.
T.H Morgan experimentally found that chromosomes are composed of DNA, Proteins and genes as specific regions of chromosomes.
Pseudo-chromosomes are found in
Prokaryotes
DNA packaging
Containment of DNA(genome) in the nucleus of eukaryotes or in the nucleoid of prokaryotes.
DNA packaging- Prokaryotes
Large circular DNA of diameter 350 micrometers is folded by forming 40-50 loop domains with the help of core proteins and RNA. due to this diameter decreases to 30 micrometers. As the 2nd step formed loop domains coils further forming super coils decreasing the diameter upto 2 micrometers,
DNase enzyme can uncoil supercoils by introducing single stranded nicks.
DNA packaging- Eukaryotes
- DNA double helix winds up around a complex of 8 histone proteins to form nucleosomes. Adjoining beads are linked by linker DNA.
- Nucleosomes twist and turn in a spiral manner to from chromatin fibers of diameter 30 nm.
- Chromatin fibers forms looped domains by attaching to a protein scaffold. Diameter about 300 nm.
- Looped domains folds and compress forming chromatids of 700 nm thickness.
Thickness of a chromosome is 1400 nm.
Euchromatin
Lightly packed chromatin which are rich in genes and involves in transcription.
Heterochromatin
Tightly packed chromatin which mostly contains inactive genes.
DNA replication
Process which copies a double stranded DNA to produce 2 identical copies.
Importance of DNA replication in evolution
Rare errors occurs in DNA replications introducing mutations which results in variations. Variations leads to evolution.
DNA synthesis occurs in which direction
5’ end 3’ end
What are the small fragments of laging strands
Okazaki fragments
What are the major enzymes and proteins involving in DNA replication and their functions
Helicase - Unwinds the double helix and separates the 2 DNA strands.
Topoisomerase - prevents further twisting of DNA molecule due to unwinding and relieves strain by introducing breaks. Relaxation of tightly wound DNA.
Primase - DNA polymerase only can add nucleotides to a already formed polynucleotide chain only. Primase forms a small fragment of RNA primer forming the DNA- RNA hybrid.
Single strand binding protein - prevents rebinding of DNA strands.
DNA polymerase - intiates DNA polymerization.
DNA ligase- seeling the gap between nucleotides.
Error of DNA polymerase
105
After proof reading 1010
2 types of DNA poymerase and their functions in the process of DNA replication
DNA polymerase 3- Adding nucleotides to the DNA template strand and extension of the new DNA strand.
DNA polymerase 1- Removal of the RNA primer and replacing it with DNA.
Differences between prokaryotic and eukaryotic DNA replication
prokaryotes usually have one Ori while an Eukaryotic
chromosome has several Ori.
The DNA polymerases in eukaryotes and prokaryotes are different from each other in their structure, while having the same functions.
The DNA replication of the prokaryotes occurs continuously, whereas in eukaryotes it happens only in the S-phase of cell cycle.
Nucleotide excision repair
Enzymes like nucleases can cut off the mismatched sequences and replace with correct nucleotides.
Gene
Fundamental physical and functional unit of heredity.
Operon
A group of genes that functions as a single transcription unit.
Consist of a promoter region, operator region and the region containing genes 1 after the other. All the genes in a operon is transcribed into 1 mRNA molecules and then translated into several proteins.
What is the transcript of a gene
pre-mRNA which undergoes processing where introns are excised and exons are joined to from mature mRNA
Gene expression
Gene expression is the process by which the information stored in a gene is used to make a functional gene product.
Final gene products
Is usually a polypeptide
However several RNAs also function as final gene products (rRNA, tRNA)
Who found out that inherited diseases are caused by the inability to produce related enzymes as a result of inborn errors in metabolism and what was this diseased condition called
Archibald Garrod
Alkaptonuria
-is due to inability to make the metabolic enzyme which metabolizes the chemical alkapton. In patients alkapton remains in urea and its oxidation results in black colouration of urine.
Genetic code
Is a triplet code where the amino acids are coded by triplets of nucleotides
Genetic code has 64 codons
Degenerative genetic code
Certain amino acids are coded by more than 1 codon
Codon
Triplet of nucleotide bases of mRNA or coding for an amino acid formation is called a codon
start codon
AUG
stop codon
UAA, UAG and UGA
Polysomes
mRNA which is being translated actively containing several ribosomes attached is known as polyribosomes or polysomes.
Protein trafficking
Signal peptide guides the polypeptide to a particular location in the cell or to be secreted
Mutation
An alteration of the nucleotide sequence of the genome of an organism
What is a point mutation
If only 1 nucleotide pair is altered due to the mutation it is known as a point mutation.
All single nucleotide pair substitutions are point mutations.
3 types of single nucleotide pair substitutions
Silent
Missesnse
Nonsense
Silent mutations
In these mutations though nucleotides in DNA and mRNA strands are altered amino acid sequence of the polypeptide remains unchanged.
Missense mutations
If a substitution change 1 amino acid in the polypeptide it is referred to as a missense mutation
Nonsense mutations
Sometimes single nucleotide pair substitution can convert a normal codon coding for an amino acid to a stop codon resulting in the premature termination of translation.
Nucleotide pair insertion
In these mutations 1 or more nucleotide pairs are added to the wild type DNA resulting in a shift in the reading frame.
Due to this missense or nonsense mutations can occur.
Nucleotide pair deletion
In these mutations 1 or more nucleotide pairs are deleted from the wild type DNA resulting in a shift in the reading frame.
Due to this missense or nonsense mutations can occur.
4 types of chromosomal mutations due to alterations in structure
deletion, translocation, duplication and inversion
Human genetic disorders by gene mutations
Colour blindness
Sickled cell aneamia
Genetic advantages of Sickled cell aneamia
Due to the presence of this abnormal hemoglobin in red blood cells changes the shape of the RBCs to curve as a sickle from its disk-shape.
Individuals having sickled cell aneamia survive malaria attacks better than individuals with homozygous wild type alleles. This is because, the malaria parasite can not survive in sickeled RBCs.
Parasite causing malaria
Plasmodium vivax
Genetic disorders of humans due to aneuploidy (chromosomal mutations)
Down syndrome
Turner syndrome
Kleinfelter syndrome
Non-disjunction
Inability to separate a pair or pairs of chromosomes in meiosis
Down syndrome
This is due to the trisomy of the 21st chromosome resulting in the presence of 47 chromosomes in an affected individual. Almost all males and half of the females with dow syndrome are sexually underdeveloped and sterile. They have short life spans but has a low risk of developing high blood pressure and atherosclerosis.
They have a high risk of developing leukemia and Alzheimer disease.
The risk of having a baby with Down syndrome increases with the age of the mother. This is caused by nondisjunction in meiosis-I.
Turner syndrome
Turner syndrome is due to monosomy in X chromosome. In very rare cases, there are females
with only one X chromosome, and hence their genotype is XO.
These individuals are phenotypically female, but they are sterile because their sex organs do not mature.
They have normal intelligence but has extra skin in the neck, puffiness or swelling of the hands and feet, skeleton abnormalities, heart defects
Kleinfelter syndrome
This is due to rare condition having an extra X chromosome in the genotype of XXY. Since it carries a Y chromosome, these individuals are males.
There testes are abnormally small. One out of the two X chromosomes is inactivated. Yet these males may have enlarged breasts and may develop other female body characteristics. They have subnormal (less than normal) intelligence.
Major steps of DNA isolation
Homogenization or disruption of cells
Inhibition of DNAse
Dissociation of nucleoprotein complexes
Removal of contaminating materials
Precipitation of DNA
Chelating agents
When the cells are broken, the DNA may get in touch with DNA degrading enzymes such as Deoxyribonuclease (DNAse). Therefore, DNA must be
protected from such enzymes causing shearing. Chelating agents are added to remove metal ions required for nuclease activity.
DNA-protein interactions are disrupted with
SDS, phenol, or proteolytic enzymes.
DNA dissolved in aqueous phase is precipitated with
cold ethanol.
The precipitate is usually re-dissolved in a buffer. RNA is removed by limited treatment with DNAse free RNAase (ribonuclease)
restriction endonuclease
The enzymes that recognises specific DNA sequences and cuts at these sites (restriction sites or cleavage sites). e.g: EcoRI
DNA ligase
Cut DNA fragments from various sources are joined together by forming phosphodiester bonds. e.g: T4 DNA ligase from T4 bacteriophage
Taq DNA polymerase
The most widely used DNA polymerase is Taq DNA polymerase. This is a heat stable enzyme isolated
originally from the thermophilic bacterium Thermus aquaticus
Reverse transcriptase
Enzymes which can make DNA on an RNA template is also very useful in gene technology. These enzymes are called reverse transcriptase. Used to make cDNA
Separated DNA in Agarose gel electrophoresis is stained by
by ethidium bromide and be visualized by exposing to UV light
most commonly used DNA joining enzyme in gene technology
T4 DNA ligase
The source of this enzyme is T4 bacteriophage viruse
Buffers
A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. Its pH changes very little when a small amount of strong acid or base is added to it.
DNA Probes
A DNA probe is a fragment of single stranded labeled DNA used to detect the presence of complementary nucleic acid sequences by hybridization.
Southern Blotting
The denatured bands on the gel need to be transferred to a nitrocellulose or nylon filter membrane by a process called Southern Blotting.
2 main methods of labeling DNA probes
By incoperating with radioactive isotopes( phosphorus 32 isotope)
By adding a florescent molecule
Recombinant DNA technology
DNA from 2 or more species are joined together and inserted into a host to obtain new genetic combination.
Major steps needed to make a recombinant DNA molecule (rDNA)
Isolation of DNA from different sources.
Restriction digestion of isolated DNA with restriction enzymes.
Seperation of DNA fragments by gel electrophoresis.
Identification of the correct fragments with desired nucleotide sequences using probes.
joining the DNA fragments from multiple sources using DNA ligase.
Vectors
vectors are vehicles to carry the DNA of interest into a host for multiplication or cloning.The vectors used in cloning of DNA are called cloning vectors.When the vector is carrying the foreign DNA, it is called a recombinant vector
Types of vectors
plasmids, bacteriophages, yeast artificial chromosomes
or YACs
Essential requirements of a DNA vector
Origin of replication (Ori-site)
Multiple cloning site
Marker genes
Antibiotic resistant genes
Commonly used as marker genes
ampicillin resistant genes, tetracycline resistant gene
They are used to distinguish between transformed and untransformed bacterial cells.
Because only transformed cells have the specific antibiotic resistant gene to survive in that particular antibiotic medium.
e.g: Bacterial cells having ampicillin resistant gene as the marker gene can survive in ampicillin medium.
DNA libraries
DNA libraries are a collection of microbial cultures each propagating a different segment of total genomic DNA cloned in a population of identical vectors.
Transcriptome
Totality of mRNA
Converted to single stranded cDNA by reverse transcriptase.
Differences between genomic and cDNA libraries
In cDNA libraries non coding DNA sequences such as intergenic DNA and introns are not expressed.
Types of DNA delivery systems
Transformation
Transduction
Gene gun
Agarobacterium mediated gene transfer
Agarobacterium causes which disease
Crown gall disease
This infection causes a tumor on the plant
Restriction Maps
A restriction map is a diagram showing the position
of each restriction site with respect to each other and the distance between these sites.
Distance is measured in units of kilo base pairs (kB)
Components present in a PCR mixture
DNA sample to be copied
Deoxy-ribo nucleotide tri-phosphates
Taq DNA polymerase enzyme
DNA primers
Buffer solution
Mg2+
3 main steps in the thermal cycle of PCR
Denaturating
Annealing
Extension
Microbiome
Is the totality of microorganisms in a particular habit including human body and different environments.
A typical PCR will have how many cycles
35 to 40
Metagenomics
Is a science in which DNA present in an environment is extracted as a community DNA and studying this sample as a whole.
Denaturation of DNA in PCR mixture is done by
Heating the PCR mixture upto 95 Celsius by using the PCR machine also known as the thermal cycler.
What are used in DNA fingerprinting
Genetic markers known as small tandem or microsatellite DNA
STR s
Small Tandem Repeats
Eukaryotic DNA contains some non-coding sequences where two to six base pairs are repeated tandemly (one after the other)
Applications of DNA Fingerprinting
Criminal identification and victim identification( forensic care)
Paternity testing
Identifying infectious agents
Heterochromatin contributes in
Gene regulation
Epigenetic inheritance
Protection from chromosomal integrity
Nondysjunction of chromosomes
Inability to separate a pair or pairs of chromosomes in meiosis
Color blindness
More common in males than in females due to mutations on 1 or more genes located on X chromosome
In humans genes coding red and green pigments are located in the X chromosome while for blue it is on the chromosome 7
Gene pollution
Spread of a foreign gene in naturally growing plants
Name the purine bases and pyrimidine bases
Purine - A,G
Pyrimidine - U,T,C
New amino acids are added to the
C terminus of the growing polypeptide chain. A peptide bond is formed between the carboxyl group in the P site with the amino group in the A site.
Monosomy and trisomy
If a gamete containing 1 less chromosome unites with a normal gamete the resulting zygote is an aneuploid(2n-1). This is known as monosomy.
If a gamete containing 1 more chromosome unites with a normal gamete the resulting zygote will carry a chromosome in triplicate.
Which type of genetic mutation is sickled cell anaemia
A single nucleotide pair substitution
Where glutamic acid of the primary structure of beta-globin is substituted with valine causing excessive folding of hemoglobin
Selective degeneration of proteins
Essential for the regularion of cellular activities
Regulatory proteins need to be rapidly degraded after their function
Agrobacterium mediated gene transfer
Agrobacterium is a soil bacterium which causes tumors in plants (crown gall disease). Cells within the tumor are transformed by Ti plasmid and the segment which is transformed to plant genome is T-DNA. Only the left and right border sequence of T-DNA is needed fir efficient transformation. Hence scientists has removed most of the bacterial genes along with virulent genes and has inserted the DNA of interest into these spaces. This is known as disarming T-DNA
DNA sequencing
Process determining precise order of N bases in a DNA molecule.
