molecular biology
TRANSCRIPT
Patricia LintonManchester Metropolitan University
It’s just so amazing – that’s what!
It’s not science fiction – it’s science fact!DNA - Encodes
all of the genetic information needed for the development and functioning of all cells.
Discipline is full of amazing facts
TRANSCRIPTION
RNA PROCESSING
DNA
RNAtranscript
3
5RNApolymerase
Poly-A
Poly-A
RNA transcript(pre-mRNA)
Intron
Exon
NUCLEUS
Aminoacyl-tRNAsynthetase
AMINO ACID ACTIVATIONAminoacid
tRNACYTOPLASM
Poly-A
Growingpolypeptide
3
Activatedamino acid
mRNA
TRANSLATION
Cap
Ribosomalsubunits
Cap
5
E
P
A
AAnticodon
Ribosome
Codon
E
The central dogma
DNA
RNA
PROTEIN
TRANSCRIPTION
TRANSLATION
DNA
mRNARibosome
Polypeptide
(a) Bacterial cell
Nuclearenvelope
TRANSCRIPTION
RNA PROCESSINGPre-mRNA
DNA
mRNA
TRANSLATION Ribosome
Polypeptide
(b) Eukaryotic cell
Uses of molecular biology – endless – revolutionised science
Medical Production of recombinant proteins for treatment of disease Determining the genetic basis of cancer – thereby improving treatment and
prognosis Diagnosis of disease – fast diagnosis of HIV/TB – infectious diseases
Agricultural pest resistance/ drought resistance/increased productivity
Industrial Manufacture of proteins, strain improvement
Forensic Used to place criminals at scene of crime or rule out suspects – important for
conviction DNA fingerprinting
Archaeology Analysis of ancient DNA in ceramic food vessels
Environmental Science Identification of species Clean-up of oil spills
(a) Tobacco plant expressing a firefly gene
(b) Pig expressing a jellyfish gene
Kary Mullis – Genius or eccentric?‘Science grows like a weed every year’
‘Each of us have things and thoughts and descriptions of an amazing universe in our possession that kings in the 17th Century would have gone to war to possess’
‘Science consistently produces a new crop of miraculous truths and dazzling devices every year’
‘I have had an encounter with an extraterrestrial in the form of a fluorescent raccoon’ ???????
You can read his Nobel lecture here:http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/mullis-lecture.html
PCR – in the dark agesPCR – in the dark ages
8 BORING hours per PCR!8 BORING hours per PCR!
95º C5 min95º C5 min
35 times35 times
55º C3 min55º C3 min
72º C5 min72º C5 min
• Individual rows of receptacles can be heated – can try lots of reaction temperatures in one ‘run’
The different steps of PCR
• Water• Buffer• DNA template• Primers• Nucleotides• Mg2+ ions• DNA Polymerase
PCR recipe
The temperature profile of a PCR cycle is controlled by the thermal cycler program which results in a near exponential increase in PCR product accumulation for about the first 30 cycles.
The 3 Os!!!!!
• Used to separate molecules based on their charge and size
• Agarose or acrylamide gels can be used to separate DNA fragments
• DNA is acidic; it migrates from the negative to the positive end of the gel
• each fragment’s migration rate is inversely proportional to the log of its molecular weight
http://learn.genetics.utah.edu/content/labs/gel/
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
Longermolecules
Shortermolecules
Gel
AnodeCathode
1
2
Powersource
– +
+–
DNA FingerprintingSteps in DNA fingerprinting:
DNA isolated from tissue sample or PCR’d up
DNA cut into fragments with enzymes DNA with different sequences produce fragments of
different sizes Can use restriction enzymes to cut either side of VNTRs
for genetic typing
Fragments separated on basis of size and visualized
Each person’s set of fragments is unique
DNA Fingerprinting – identification of the remains of the Russian Royal Family
DNA fingerprinting showed that 9 different people were buried in the Ekaterinburg grave.
Romanovs would be more similar in pattern to each other than to non-relatives.
All of a child’s bands must be present in one or both of the parents.
7.6 DNA FingerprintingAdult1
Adult2
Adult3
Adult4
Adult5
Adult6
Child1
Child2
Child3
DNA FingerprintingTo see if parents and their children were Romanovs,
DNA fingerprints were prepared for relatives of tsar and tsarina.
Adult male skeleton (related to the children) was related to George, the tsar’s brother.
Adult female skeleton (related to the children) was related to Prince Philip, the tsarina’s grand-nephew.
Conclusion: the grave contained the tsar, tsarina, three of their children, and four servants.
Making your own equipment