Basis of genetics (Mendel)
Law of Segregation: When any individual produces gametes, the copies of a gene separate so that each gamete receives only one copy. A gamete will receive one allele or the other.
Law of Independent Assortment: Alleles of different genes assort independently of one another during gamete formation.
Basis of genetics (Avery, McLeod)
Experiments using pneumococcus.
Pneumococcus exists in two forms:
R-form ("rough"), lacks capsule that protects it from immune systems, non-pathogenic.
S-form ("smooth"), contains capsule that prevents immune system from destroying it, consequently pathogenic
(1) Bacteria that were infected by non-pathogenic R form were cultured.
(2) Bacteria infected with the S-form were heat-treated (killed). The solution was divided into 3 aliquots. Each was treated with a different enzyme:
a. protease (e.g., trypsin)
(3) The culture of live, R-form (non-pathogenic) infected bacteria was divided into three aliquots, with one added to each enzyme-treated aliquot (step 2)
(4) Protea and RNAase treated solutions were able to transform the R-infected cells to the S-form. The DNase-treated cells were not capable of changing the R to S.
Conclusion: the transformation from R to S was DNA mediated.
Know the following?
Simple transition from diploid to haploid followed by fertilization (regenerating a diploid cell).
Meiosis consists of two division one reductional (di to haploid) and one equational (haploid to haploid).
Know Punnet square info?
Structure of DNA
Right-handed double helix maintained by H-bonds between the bases (internal).
The phosphate backbone faces the outside of the double helix.
2nm in diameter, bases separated by 0.34 nm.
5' to 3' anti-parallel.
A-T and G-C base pairing (Chargaff's Rule).
Specific differences between A, B, and Z forms.
* Shallow major wide groove, narrow and deep minor groove.
* Most common form of DNA.
* Right-handed, 10.4 nucleotides per turn
* Bases inside, phosphate outside.
* Major groove and minor groove are about the same depth and width.
* Second most common form of DNA (DNA-RNA or double-stranded RNA molecules are mostly seen as A-form)
* Right-handed, 11 nucleotides per turn
* Bases inside, phosphate outside.
* Single narrow deep groove
* Unusual form of DNA
* Left-handed, 12 residues per turn
* Bases are pointing outward and phosphates are inside the double helix.
* Often present when DNA displays high GC content. Flipping can be triggered by supercoiling.
Replication models (various models and proof of concept).
Conservative: The parent strands serve as a template for the generation of an entirely novel second DNA molecule. The parents remain together after replication.
Dispersive: Post-replication DNA strands are made of some parent segments and some novel segments.
Semi-conservative: In each daughter DNA molecule, there is one parent strand one novel strand.
Experiment based on bouyency by Meselson and Stahl (1958).
DNA labelled using N-14 and N-15, as bouyency of DNA containing N14 is different from that containing N15. Therefore, they can be separated by centrifugation.
Bacteria first grown in presence of N15 incorporate heavy isotope. After incorporation, they were transferred to N14-containing media (light isotope). First replication (20 minutes) generated a single band midway between expected locations for an entirely N14 DNA molecule and an entirely N15 DNA molecule. This is consistent with either semi-conservative (each daughter DNA molecule made of one N14 strand, one N15 strand) or dispersive models (each daughter DNA molecule made of 50% parent, 50% novel).
Second replication after 40 minutes: Yielded two bands, one (again) mid-way between purely N14 and purely N15, one at purely N14 location. This was consistent with semi-conservative model only.
DNA codes for RNA
Transcription requires enzyme RNA polymerase and nucleotides. One coding strand and one non-coding strand.
Concept of codon and anti-codon
Codon: A sequences of three nucleotides that specify which amino acid will be added next during protein synthesis.
Anticodon: a unit made up of three nucleotides that correspond to the three bases of the codon on the mRNA. Each tRNA contains a specific anticodon triplet sequence that can base-pair to one or more codons for an amino acid. For example, the codon for lysine is AAA; the anticodon of a lysine tRNA might be UUU.
RNA codes for proteins
Mutation experiments on the rII gene of bacteriophage T4 (A bacterial virus), consisting in the addition of one, two or three nucleotides in the mRNA coding for a particular protein
The genetic code is the set of rules by which information encoded in genetic material (DNA or mRNA sequences) is translated into proteins (amino acid sequences) by living cells.
Various types of RNAs: structure and role.
mRNA code for protein translation. rRNA are also required to form the ribosomes (nucleoprotein complexes). tRNA needed to bring the amino acids in the proper position within the ribosome to support translation elongation.
Recombinant DNA: use of various enzymes and other molecular biology tools
Restriction enzymes: molecular scissors. Different types of RE exist. Not all of them are useful for molecular biology purposes.
They are isolated from bacteria and the name reflects the organism from which they have been initially purified (Eco R1 comes from E. coli) and recognizes GAATTC.
Certain enzymes are DNA methylation sensitive (at GC where the C can be methylated).
Can run fragments on gel. Stain with EtBr or SYBR green (intercalates between the two strands); excite with UV light.
cDNA cloning is used to clone coding sequences into a DNA vector that will be later inserted into bacteria or other organism to amplify the DNA. First the mRNA will be isolated. mRNA end with a poly-A sequence that will hybridize with a poly-dT. Oligo dT will be attached to beads and used to collect the mRNA. After release from the beads, the mRNA is reverse transcribed (DNA from RNA) and a double-stranded DNA molecule with the same sequence as the initial mRNA is created. It is ligated to a vector (after addition of linker that will have a sequence complementary to that of the vector (for proper base pairing), then ligated to form a cDNA clone.
To allow cloning, once small linear DNA fragments have been generated the molecules need to be re-attached. DNA ligase will re-attach DNA fragment that have complementary base pairing or blunt-end fragments. DNA ligase requires ATP to regenerate a phosphodiester bond on each strand.