Molecular Biology

Molecular biology                                                                                            

 is the study of biology at a molecular level. The field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry.

It involves the understanding of how molecules give rise to biological characteristics from a string of bases to living organisms such as

– DNA replication, transcription, translation

– Genes, gene organisation, non-coding regions, phenotype

– Link between chemistry and biology

Prokaryotic genomes

  Generally contain single circular replicons

 Not strictly chromosomes – no chromatin

 Supercoiling

  Some have multiple chromosomes

  Some have linear chromsomes

 Often extra chromosomal DNA present

Plasmids

Very useful for DNA manipulation

Eukaryotic genomes

Eukaryotic genomes are larger than those of prokaryotes.

Much of eukaryotic DNA is noncoding.

Even the coding regions of genes contain sequences that are not translated.

Eukaryotes have multiple chromosomes.

Each eukaryotic chromosome has a single, continuous, double helix of DNA.

Nucleic acid extraction                                                                                        

• Disrupt cell • Stop enzymes • Remove proteins / fats • Purify nucleic acids • Separate types of nucleic acid • Concentrate sample

 DNA Extraction - variations

• Phages / viruses– Need to purify from host cell • Plasmids– Separate DNA according to size (CsCl / alkaline lysis)

• Whole chromosome extraction – Gentle extraction, limited pipetting, shaking etc. • Plant cells – Need to disrupt cell wall (nitrogen / grinding / CTAB)

• Dirty prep– Chelex / hydroxyapatite / purification columns

After extracting Dna......                                                                                            

Reconsider history

– Function as transforming material – Look at Basic structure

– Look at sections – Amplify sections – Read individual bases

Restriction / Modification

• A primitive bacterial immune system • Enzymes that ‘restrict’ infecting phage

from growing by cutting DNA • Enzymes that ‘modify’ own DNA to prevent cutting

Cloning                                                                                                                 

• Cut DNA with restriction enzyme and Add extra DNA cut with same enzyme

• Allow DNA to anneal – creation of genetically modified DNA

• Insert into organism – GMO created

 Types of Cloning

• Vectors developed for other organisms – YACs - yeast artificial chromosomes

– Ti plasmid to transform plant cells – Viral replicons for animal cells

• Used extensively in gene function research, biotechnology, genome sequencing, etc.

Amplification                                                                                                        

• Always a drawback of amount of material for studies    • Development of PCR

– Literal recreation of replication in a tube

• Allowed work from small samples (forensics) • Allowed purification

 Understanding the code has allowed…

 – the diagnosis of genetic disease

 – recombinant DNA technology

 – biotechnological production of cloned products

 – understanding the mechanisms underlying the expressions of genes

Mitochondria                                                                                                     

• Ancient symbiosis between eubacteria and eukaryote ancestor

• Mitochondria retain separate bacterial type

genome – mt DNA – and ribosomes

– Encodes key functions for expression of electron

transport proteins

• Much reduced size (compared to bacteria) –most genes in nuclear genome

• 2-10 copies per organelle

Inheritance                                                                                                             

• At fertilisation, sperm mitochondria are not

established in the zygote – Actively eliminated

• mtDNA inherited in the maternal line only

• Determination of DNA sequence allows tracing of maternal inheritance

• Mitochondria

– Circular DNA - 20Kb to 2.5Mb

• Plastids (including chloroplasts)

– Circular DNA - 75Kb to 250Kb

• Viruses

– RNA / DNA, circular / linear, single stranded /

double stranded – 2Kb to 1.2Mb

• Transposons / Insertion sequences /

Ribozymes (cf. prions

Advantages of mtDNA                                                                                          

• 1% of cellular genome is mtDNA

– Multiple mitochondria

– Multiple mtDNA copies in each

• mt genome is small but has high copy

number

• In decayed, degraded or burnt tissue

mtDNA is far more likely to survive than

single copy DNA