molecular biology

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