molecular biology Flashcards
what are nucleotides?
monomeric units from which nucleic acids are built
(base + sugar + phosphate)
involved in nearly all biochemical processes
- cellular energy exchanges (ADP, ATP)
- coenzymes (NAD, FAD, CoA)
- second messengers (cAMP)
what are nucleic acids?
- deoxyribonucleic acid- DNA
- ribonucleic acid- RNA
functions:
- direct synthesis of proteins
- transmit genetic information
what is a nucleoside?
base + sugar (NO PHOSPHATE!!)
what is the difference in DNA and RNA?
DNA- deoxyribonucleotides
RNA- ribonucleotides
what is ATP?
- main biological energy store
- produced in mitochondrion
what is cAMP?
second messenger in action of many hormones
- Hormone binds to receptor in plasma membrane of cell
- Adenyl cyclase stimulated
- ↑ Adenyl cyclase activity leads to ↑ cAMP inside cell
- cAMP acts inside cell to alter rate of one or more processes
what is the structure of DNA?
- Found mainly in cell nucleus
- DNA backbone consists of deoxyriboses linked by phosphodiester bridges
- Sugar of one nucleotide joined at C-3′ (OH) to phosphate group attached to C-5′ of sugar of the next nucleotide (3′-5′ phosphodiester bonds)
- Bases project from sugar residues
what bonds hold together the two strands in the DNA double helix?
hydrogen bonds
what is the general structure of RNA?
Found mainly in cytoplasm
Same 5′─3′ backbone as DNA
RNA differs from DNA:
Uracil (U) replaces thymine (T)
Ribose replaces deoxyribose
Single-stranded (no base pairing)
what is a hairpin loop?
local double strand formation can occur within RNA
what is the genetic code transmitted via?
genes
what is the genome?
whole genetic information within a single cell nucleus
what are the steps of protein synthesis?
- In nucleus, DNA unfolds (H bonds split) to expose base pairs of template strand
- Transcription: RNA polymerase copies sequence of bases in template strand of DNA to produce mRNA
- mRNA translated by tRNA, using ribosome as a functional support (in cytoplasm)
describe translation
Ribosome: small and large subunits, contain proteins and rRNA
tRNA:
- Carries each amino acid to ribosome (one specific tRNA for each amino acid)
- Contains anticodon (3 bases) complementary to sequence of 3 bases (codon) on mRNA
- Amino acid transferred to growing polypeptide chain
- Sequence of mRNA codons determines sequence of amino acids in a protein
how many amino acids exist?
20
what is a mutation?
a random permanent alteration in a DNA sequence
caused by:
- Errors in DNA synthesis that can occur spontaneously at low frequency
- Chemical mutagens
- Ionising radiation
describe the different possible effects of a substitution mutation?
- Conservative mutation: amino acid is replaced by one with similar properties
- Non-conservative mutation: amino acid is replaced by one with different properties
- No mutation: in many cases a change in the third position of the codon does not change an amino acid
what does insertion/deletion of a base?
Causes a frame-shift, and hence synthesis
of a protein that has a completely different
sequence downstream of the mutation:
If any of these changes introduces or removes a stop codon → premature or delayed termination
Sickle-cell anaemia is caused by a single-base substitution (GAG to GTG) in the β-chain of haemoglobin. This replaces glutamic acid with valine, which is a non-conservative mutation
More drastic mutations may also occur eg.
deletion or duplication of longer stretches of
DNA
what is recombinant DNA technology?
DNA from different organisms is “cut and pasted” together, producing recombinant DNA
why is recombinant DNA technology important?
- Vaccine production
- Protein therapies:
- human insulin
- human growth hormone
- interferon
- Production of blood clotting factors to treat haemophilia
- Gene is cloned into a plasmid which is then introduced into a bacterial cell; bacteria produce the protein, which is then purified and used in patients
what is gene therapy?
- Replace faulty (mutated) gene with a healthy one or add a new gene into the genome
- Used to treat or prevent disease e.g. cancer, diabetes, heart disease, cystic fibrosis, haemophilia
what is gene cloning?
- Produces large numbers of copies of a particular piece of DNA
- Genes are usually cloned by isolating them using restriction enzymes, followed by gel electrophoresis and inserting them into a plasmid
- Plasmid is then introduced into a bacterium, and the bacterium allowed to grow to produce large numbers of cells and hence many copies of the gene
- The gene can then be re-isolated using the same restriction enzyme
what are restriction enzymes?
- Cut double-stranded DNA at specific DNA sequences
- These sequences are typically 4 to 6 base pairs in length and “palindromic” ie. they read the same in both directions
- Most restriction enzymes make a staggered cut (“sticky ends”), which allow DNA fragments to re-associate by base pairing
- After re-association, the fragments can be rejoined by DNA ligase
describe gel electrophoresis
- Used to separate DNA fragments on basis of their size
- Samples are applied to a gel immersed in buffer and a current is applied
- Negatively-charged DNA migrates from the negative electrode (top of gel) to the positive electrode (bottom of gel)
- Larger DNA fragments migrate more slowly than smaller DNA fragments
describe the procedure of gene cloning
- To insert a gene into a plasmid, a restriction enzyme is chosen that cuts on either side of the gene but not in the middle
- The gene is separated from other DNA fragments by gel electrophoresis
- A suitable plasmid is linearised (cut at one point) using the same restriction enzyme
- The cut plasmid and gene are mixed, and the sticky ends of the plasmid and gene are allowed to “anneal” (associate by base pairing)
- The annealed ends are covalently joined using DNA ligase
- The plasmid, now containing the gene of interest, is introduced into the host bacterium
- The bacteria are grown into a colony, using antibiotic resistance genes in the plasmid to select colonies containing plasmids
- Cloned cells are lysed and the plasmids isolated by centrifugation
- Plasmids are cut with the restriction enzyme, releasing the cloned gene
what is Sanger sequencing?
- Also known as the dideoxynucleotide chain termination method
- Remains in wide use, generally for small-scale projects e.g. single genes
- Synthesis of new DNA strands complementary to a single-stranded template strand in vitro
what is a primer?
short strand of bases, serves as a primer for synthesis of the complementary DNA strand by primer extension
what is the function of DNA polymerase?
enzyme that catalyses DNA strand synthesis
describe chain termination
- Interruption of DNA strand synthesis depends on presence of ddNTPs
- 3’ OH group of dNTPs replaced by H in corresponding ddNTP
- ddNTPs incorporated into growing DNA chain (as the final nucleotide) but lack 3’OH required to form a phosphodiester bond with next nucleotide → chain termination
- Synthesis is interrupted at every possible site in a given population of molecules, resulting in hundreds of DNA fragments of varying length
- ddNTPs are added at a much lower concentration than the standard dNTPs (ratio of 1:10 to 1:300) to allow strand elongation sufficient for sequence analysis
what is the procedure for DNA sequencing?
- DNA to be sequenced is mixed with primer
- Primer binds to 3’ end of DNA
- DNA-primer mixture divided into four separate reaction tubes containing:
- all four dNTPs
- one of the four ddNTPs (A, C, G, T tubes)
- DNA polymerase
- Chain synthesis proceeds in each of the four reaction mixtures
- Gel electrophoresis separation of reaction products → band corresponding to each position of chain termination appears
- DNA bands detected by autoradiography (35S) or by laser in an automated sequencer (IRD800)
- DNA sequence can be deduced from the pattern of bands in the four lanes:
- a dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of a ddNTP
- the terminal nucleotide base can be identified according to which ddNTP was added in the reaction giving that band
- the relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence.
what is high throughput sequencing?
- Also known as next generation sequencing (NGS)
- No need for cloning, highly scalable
- Sequence millions of genes and entire genomes at once
- Cheap and rapid
- Requires substantial bioinformatics analysis
- Key platforms: Illumina (HiSeq, MiSeq), Roche 454, Ion Torrent (PGM, Proton)
