Here is a complete set of organised notes for Lectures 1-8 of BIOC0005 Molecular Biology. This course is taken by UCL year 2 students studying Molecular Biology, Biochemistry and other Life Sciences degrees. I received first honours and was on the Dean's list, in which my main study method was by m...
Central Dogma
• Explains the ow of genetic information in a cell (DNA -> RNA -> Protein)
• Experiments show that there is also RNA replication (usually people think of DNA replication) and reverse
transcription of RNA back to DNA (RNA retroviruses for example.)
◦ Francis Crick recognised the possibility of these in his paper.
• However, there is no study showing protein can go back to RNA or DNA.
DNA
• Unbranched anionic polymer made of 4 di erent types of nucleotide
◦ Anionic - phosphate group
• deoxyribose sugar has no OH (hydroxide group) at 2' carbon.
• Be able to recognise the bases!
• Nucleotides are linked by 3' and 5' carbons
• Directionality: biological DNA synthesis is always from 5' to 3'; (chemical synthesis is from 3' to 5').
• Note how condensation occurs in the picture
DNA base pairings and stacking
• Double strands are held together by hydrogen bonds
between base pairs.
• Purine to pyrimidine pairing allows distance to be
equal
◦ Important because phosphate backbone is
negative, too close = repulsion
◦ If too far, then the next base pairs are too far to
form hydrogen bonds.
• Base stacking cause the DNA to twist into a double
stranded helix
◦ Base are hydrophobic - energetically
favourable to stack.
DNA structure types
• B form, A form (dehydrated DNA), Z form, triple
helical DNA (introduce third helix to b-DNA) exist at di erent conditions
• Usually nature is right handed helix
B form DNA
• 10.5 bp per turn
• Rise of 3.32nm
• 2.0 nm in width
• Have major and minor grooves
Asymmetry of DNA - idea that L and Width ratio is really big (way longer than width)
Post-synthesis DNA modi cation - methylation is very common
• Methylation of adenine of cytosine
◦ Adenine -> N6-methyladenin
◦ Cytosine -> N4-methylcytosine
• Methylation can
◦ Change activity of DNA segment without changing the actual sequence ie. rate of transcription
◦ Mark a DNA strand as parental strand during replication (ie. methylated is parent strand, so after replication
can correct errors (if any) on the newly synthesized non-methylated strand. Recall semi-conservative
replication)
RNA
• Base of Thymine (in DNA) is replaced by Uracil that lacks methyl group
◦ Reason: in early evolution, we used RNA instead of DNA. RNA had AUCG. Cysteine spontaneously
deaminates into uracil. So when DNA took over, it switched uracil to thymine so that it can still
, di erentiatetures. which uracils came from cysteine.
• RNA (think mRNA) are single stranded but can also fold to form local double stranded secondary structure
• Ribose sugar has extra OH group at the 2' carbon, this increase repulsion between the two complementary
strands (compared to DNA)
• Many types of RNA with di erent structures ie. clover shaped tRNA
Proteins
• Unbranched polymer of L amino acids joined by amde bonds
• Synthesis is from N terminal to C terminal. (There is directionality)
• 20 di erent amino acids
• 2 extra proteinogenic amino acids: selenocysteine and pyrrolysine
◦ Get incorporated into certain proteins under speci c conditions
◦ These non-standard amino acids do not have a dedicated codon, but are added in place of a stop codon
when a specific sequence is present,
• Order of amino acids determined by mRNA! (Not DNA, because 1. Introns 2. RNA editing: editing of RNA after
DNA transcription)
• Protein splicing: change the linear information in a polypeptide chain
Lecture 1 Continued Molecular tools and techniques
Nucleic acid analysis
• Absorbance to quantify DNA and RNA
◦ DNA and RNA absorbs at UV region (below 290nm)
◦ Ring structure of the bases (dNTP and NTP) absorb UV
◦ Maximum absorbance at around 260nm.
◦ 1 A260 unit for dsDNA = 50ug/ml
◦ 1 A260 unit for ssDNA = 37ug/ml
◦ Usually there are more RNA than DNA in cells so RNA can contaminate the DNA signal.
◦ Protein will also absorb at 280nm.
◦ pure DNA sample A260/A280 ratio ~ 1.8.
• Absorbance to look at hyperchromicity
◦ Double stranded DNA melting to ssDNA increases absorbance in real time.
◦ Tm is the melting temperature (midpoint). More GC content = higher DNA TM
• Hybridization is used to look at complementarity of sequences and if organisms are related
◦ dsDNA -> heat -> ssDNA -> hybridise to see
• Gel electrophoresis
◦ Negatively charged DNA migrates through pore.
◦ Linear DNA = migration rate proportional to length
◦ Circular DNA- depend on conformation
◦ Higher Percentage of agarose increase resolution. Use higher percentage for smaller fragments.
• EtBR staining visualises DNA and RNA
◦ EtBR planar structure intercalates between base pairs which is a hydrophobic environment.
◦ UV light re-emitted as orange uorescence.
◦ EtBr in aqueous gel is weak because of quenching.
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