BIO2015: Lecture 2

Essential Techniques in Molecular Biology; recombinant DNA technology

DNA extraction:

• DNA in nucleus of cell- must access/isolate DNA without damaging
• 1) cell membrane is disrupted with detergent, for stronger method- urea based chemicals, another way is freeze/thaw cycle
• 2) alcohol added to tube to separate DNA from other cell components because it won't dissolve in it- DNA forms precipitate
• 3) DNA spooled onto glass pipette

DNA can be extracted from any nucleated cell, DNA must be extracted as high purity for downstream applications

purity of DNA can tested using spectrophotometer:

• DNA conc in ug/ml = OD at 260nm x dilution factor x 50
• the ratio of absorbance at 260 and 280 A260/A280 gives indication of DNA purity, high quality DNA= 1.7 to 2.0

nucleic acid absorbs at 260nm whereas proteins absorb at 280nm, reading of 1.7 to 2.0 indicate high nucleic acid and very litle protein which is good

Commonly used enzymes in molecular biology:

Difficult to physically manipulate DNA, must use enzymes

can use them to cut, manipulate DNA strand itself, can join DNA together- do this in controlled/reproducible manner

Cutting/joining is most famous example

Once pure sample has been prepared, next step is to construct recombinant DNA molcule

manipulations are performed in vitro- i.e. in test tube

almost all techniques require purified enzymes, these exist in bacteria and had natural role in host e.g. DNA replication, transcription, breakdown of unwanted or foreign DNA (virus), repair of mutated DNA and recombination between different DNA molecules

after purification from cells extract, many of these enzymes can be persuaded to carry out their natural reaction, under artificial conditions

The range of DNA manipulative enzymes:

• 5 broad classes- depends on reaction they catalyse
• Nucleases- cut, shorten, or degrade nucleic acid molecule
• Ligases- join nucleic acid together
• polymerases- make copies of molecules
• modifying enzymes- add or remove different groups onto/from DNA
• topoisomerases- introduce or remove supercoils from covalently closed circular DNA

Nucleases:

• degrade DNA molecule by breaking phosphodiester bonds that link one nucleotide to next in DNA strand
• 2 types of nucleases:
• 1) exonucleases: remove nucleotide one at a time from the end of DNA molecule (e.g. Nuclease BAL3, E.Coli exonuclease III etc
• 2)Endonucleases: break internal phosphodiester bonds within DNA molecule (e.g. S1 nuclease, Mung Bean Nuclease DNAsel, RNAse A, restriction enzyme etc.)

RNAses:

• RNAseA- endoribonuclease- generic non-specific- will attack single stranded RNA, not DNA- useful because you can remove all RNA from a sample will not attack DNA at all- useful for making genomic DNA libraries
• RNAseH- endoribonuclease- doesn't attack single stranded RNA- digests RNA of RNA-DNA hybrid- bacteria sees it as foreign- specific use for making cDNA libraries

Ligases:

• repairs single stranded break in double stranded DNA molecule
• equally as usefully- can repair double stranded break- we can therefore also join 2 pieces of DNA together
• facilitated by molecule with cohesive/sticky ends but can also facilitate bonding of blunt ends
• Modes of action: catalyzes formation of phosphodiester bond between adjacent 3'-OH and 5'-P termini in DNA
• ligation of complementary sticky ends is…