Nucleic acids and chromosomes Flashcards
Components of nucleotides
Base, sugar, phosphate
Components of nucleosides
Base, sugar, no phosphate
Structure of DNA/RNA
Chain of nucleotides linked by phosphodiester bonds
3’ OH of sugar linked to phosphate group attached to 5’ carbon of next nucleotide
First nucleotide in sequence has free phosphate attached to 5’ carbon
Last nucleotide in sequence has a free hydroxyl group attached to the 3’ carbon.
Direction of DNA/RNA writing
5’ end to 3’ end
Features of the double helix
- 10 base pairs per helical turn
- Major and minor grooves
- Hydrogen bonds between bases on the two strands give helix stability
- Chains are antiparallel (they run in opposite directions)
- Negative charges on phosphate group make the sugar-phosphate backbone run on the outside with the bases on the inside.
DNA bases
Adenine
Thymine
Cytosine
Guanine
RNA bases
Adenine
Uracil
Cytosine
Guanine
Purine bases
Adenine
Guanine
Pyrimidine bases
Thymine
Cytosine
Uracil
Difference between purine and pyrimidine
Purine = double ring Pyrimidine = single ring
Watson-Crick base pairs
- Adenine with Thymine/Uracil = forms two H bonds between bases
Cytosine with Guanine = forms three H bonds between bases
Conditions used for denaturing/melting (separation) of strands
Heat
Low salt
Conditions used for re-annealing of strands
Cool
High salt
Process of hybridisation in NA analysis
- Target DNA from agar plate/gel immobilised on solid support
- DNA then readily binds single-stranded nucleic acid (denatured DNA/RNA)
- Hybridised with solution of radioactively or fluorescently labelled probe
What is hybridisation
A method for detecting specific nucleic acid sequences, homologous single-single stranded DNA/RNA molecules combine to form double-stranded molecules. Probe used to show sequence presence.
What is PCR
Polymerase Chain Reaction, an in vitro (outside of the cell) method which allows the selective amplification of a specific target DNA within a mixed collection of DNA sequences.
Process of PCR
- 2 primers designed to be complementary to target sequences
- Primers annealed to denatured DNA by lowering the temperature
- TAQ polymerase and free nucleotides (dNTPs) extend the chain
- Denature and repeat the cycle several times to generate many copies of target DNA
Temperatures for denaturing, annealing, and extension in PCR
- Denaturing = 94 degrees
- Annealing = 50-60 degrees
- Extension = 72 degrees
What types of restriction enzymes are used and what do they do
Type 2 restriction endonucleases which cleave the DNA at specific recognition sequences
What types of ends do restriction enzymes produce and what is the difference between them
Blunt ends - both strands cut at the same nucleotide.
Sticky ends - Each strand cut at different nucleotides, two nucleotides apart. Ends overlap.
Association between recognition site length and occurrence frequency in DNA
The longer the recognition sequence, the less frequently it occurs in DNA
What is a nucleosome
Structural unit of human chromosomes, consists of length of DNA coiled around a core of histones.
Packaging of DNA
DNA wrapped around histone proteins. These are positive proteins which interact with negative phosphates on the DNA. 10000 fold shortening due to histones.
What is a karyotype
An organised profile of a person’s chromosomes
