Lecture 1 – DNA EXTRACTION, SPOOLING & QUANTIFICATION

 DNA
o replication of genetic material, protein production
o Watson & Crick – double helix, 2 anti-parallel strands (spiral ladder)
 2 strands – backbone (sugar & phosphate), rungs – bases (ACTG)
 A-T – 2 H bonds
 C-G – 3 H bonds
 DNA Spooling
o Extracted using aqueous buffer soln to cells
o Lysed & chromosomal DNA is released
o DNA is soluble in water but not alcohol
o To purify: precipitate with alcohol in presence of a salt
o Amount of DNA spooled α size of DNA fragments
 Fragments are larger than AAs & sugars
o Isopropyl has a lower density than water – forms a 2nd layer above DNA sol.
o Spooling rod separates DNA from solution
o ACTG have a max. absorption @ 260nm
 > light abs. by sample, higher the [nucleic acid]
50 µg DNA /mL
o [DNA] (µg/mL) = OD260 x dilution factor x
OD 260 unit
o Low temp. protects DNA – slows down the activity of degrading enzymes
 Chilled alcohol inc. DNA precipitation rate
 Lab:
o Higher the [nucleic acid] -> more light adsorbed

Lecture 2 – TRANSFORMATION OF E. Coli WITH pGAL PLASMID

 Bacterial transformation – uptake of outside DNA by cells, a new, stable, heritable

genetic trait is acquired & integrated into bacterial DNA
 Plasmid – small, circular dsDNA that exists external to main bacterial chromosome
o Replicates independently
o Doesn’t integrate into bacterial chromosome
o Usually have genetic advantages
o Has its own machinery to clone, transfer +/ manipulate genes
 Lac operon: lac L, operator, lac Z, lac Y, lac A
o When lactose is present: repressor can’t bind to operator – lac mRNA prod
o When lactose is absent: repressor binds to operator – lac mRNA isn’t prod
o We used LacZ-; pGAL plasmid had complete lacZ gene but no MCS
 => repressor can’t bind -> lac mRNA prod
 o When lactose is absent: repressor binds to operator – lac mRNA isn’t prod
 Competency – when a bacterial cell can take up beneficial DNA & transform
genetically
o To prep competent cells:
 Treat with metal-chloride (Mg/Rb/Ca) salt
 Heat shock (rapid hot/cold)
 Place cells in an electric field
^ affect cell structure & permeability for DNA to enter
o Satellite colonies:
 => not Ampr & haven’t transformed with pGAL
 => beta lactamase diffused & inactivated Amp
 # of satellite colonies inc. if [Amp] is low or plates have been over-
incubated

Lecture 3 – Isolation & Quantification of Plasmid DNA from Bacteria

 Plasmid prep:
o Preparation & clearing of lysate
 Resuspend cells
 Done with P1
o Has RNase A & LyseBlue
 LyseBlue – color handling mech. to control for
errors. Optional, not necessary
 Alkaline lysis of bacteria (blue)
 P2
o Alkaline lysis buffer
o Has NaOH/SDS in the presence of RNase A
 SDS – makes phospholipid & protein of cell
membrane soluble -> lysis -> cell contents
released. Alkali conditions denature
chromosomal & plasmid DNA; proteins too
 Lysate neutralized, adj. to high salt binding conditions
 N3
o Colourless, indicates that SDS from lysis buffer has
been properly precipitated
o High [salt] α precipitation of denatured proteins, cDNA,
cell debris & SDS upon centrifugation
o Smaller pDNA renatures correctly in solution
o Must be mixed for precipitation but never vortexed
 Centrifuged, debris removed, clear lysate gleaned
  pDNA separated from cDNA via co-precipitation w/ insol.
complexes c salt, detergent & protein
 pDNA in clear supernatant
 During lysis: vigorous treatment -> sheared chromosome ->
free cDNA fragments in supernatant
o Adsorption of DNA into silica membrane
 Removal of RNA, cell proteins & metabolites
 High salt buffer used
o Washing & elution of pDNA
 Removes endonucleases & salts
 pDNA eluted from membrane using low-salt buffer or water
 pH dependent (max 7-8.5)
o Quantification
 UV spec – ACTG have max abs of 260nm

Lecture 4 – Restriction Endonuclease Mapping of Plasmid DNA

 Restriction endonucleases – recognize & cut a short specific palindromic DNA

sequence & cleave it from inside
o Cleaves both strands of dsDNA @ specific sequences (recognition site)
 Cleavage site located within/near recognition sequence
o EcoRI recognition site: 5’…GAATTC…3’
 Sticky end cutter (leaves hanging bases)
 Value of DNA Mapping:
o DNA fragments can be sequenced
o DNA sequences can be compared w/o specific knowledge of their nucleotides
 Separating DNA fragments:
o Separated acc. to size & shape via agarose or polyacrylamide gel
electrophoresis
 Gel – used to det. the size of DNA mols; acts as a molecular sieve
o DNA has net -ve charge (bc PO4)@ neutral pH => migrates to +ve electrode
when current is applied
o Duplex DNA detected by staining w/ intercalating substance b/w stacked bps
 Ethidium bromide
 Acridine orange
 Proflavin

Lecture 5 – PCR Primer Design

 PCR
o isolation of pieces of DNA of a known gene
 o amplification from template DNA
 helps wrt DNA identification & manipulation
 detection of pathogens (usually viral)
 detection of genetic variations
o doesn’t use a living organism
o steps (x30-40):
 denaturation – 94 deg C
 dsDNA -> 2 ssDNA
 annealing of primers – 60 deg C
 complementary regions of ssDNA
 extension/synthesis – 70 deg C
 DNA Pol
 Primers
o Short ssDNA mols – 17-25 nucleotides
 Long enough for specificity
 Short enough for easy binding & annealing temp
o Bind to specific DNA sequences
o Initiation sites for DNA polymerization
o Bad primers:
 Little/no PCR product
 Amplify unwanted DNA fragments
o Complementary to target sequence alone ensures taq Pol only copies target
region
o Usually two – forward primer (L->R) [5’->3’] and reverse primer (R->L) [3’->5’]
 Only first round of PCR – longer than unit length strands occur
 After 18-20 cycles taq Pol loses efficiency and [dNTPs] decreases
 Melting temperature, TM – temperature @ which ½ of DNA duplexes dissociates
from dsDNA to ssDNA.
dsDNA < TM < ssDNA
o 52 deg C – 58 deg C – perfect primer
o > 65 deg C – secondary annealing
o NB: annealing is usually 5 deg C below TM
o Annealing temp too high – primer won’t anneal to target DNA
o Annealing temp too low – primer will anneal to wrong sequences that aren’t
100% compl.
o For 2 primers: TM should be within 5 deg C of each other. < 5 – ideal
o Increased [GC] ṝel. to increased TM
 Wallace Rule
TM (deg C) = 2(A+T) + 4(C+G)
  If primers are self-complementary then they’ll self-anneal & won’t bind to target
DNA.
o Hairpins
 Intramolecular base pairing of one primer; ideal < 3 IM bps
o Primer dimers
 2 different primers binding
 Bp between forward & reverse primers – heterodimer
 Self-dimer if 1 dimer bps

Lecture 6 – VNTR

 Polymorphic DNA – chromosomal regions that differ from one person to the next
 VNTR – 15-70 base pair sequence tandemly arranged, repeated 5-100x
 STR – VNTR but only 2-4 nucleotides long
 DNA fingerprinting
o CODIS, 1990 – FBI forensic crime database
o Steps:
 Collect sample
 Treat it – lyse cell membrane to obtain DNA
 Amplify VNTRs & STRs by PCR
 Separated & examine amplified DNA via agarose gel electrophoresis
 Famous VNTR: D1S80 – chr. 1, 16 nucleotide sequence repeated 16-40x
o D1S80 homozygous – equal repeat #s on both homologues of chr 1
 single PCR product after gel analysis
o heterozygous – repeat numbers differ
 2 distinct PCR products
 Method:
o Collect sample
o Treat sample with detergent – lyse cell membrane & obtain DNA
 Lysis solution – 25mM Tris-HCL & 5% chelating agent
 Chelating agent removes Mg (needed by DNA polymerase)
o Amplify by PCR
 Taq polymerase – stable @ high temperatures
 1. Denaturation – 94 deg C
 2. Annealing – 40 – 65 deg C
 3. Extension 72 deg C
o Separate & examine by agarose gel electrophoresis
 Lysis solution c Tris-HCl
o Chelating agent
 Sequesters Mg from nucleases
  Boiling helps with cell lysis, doesn’t denature DNA
o Nucleases don’t degrade bc Mg trapped
 Proteinase K
o From Tritirachium album – cleaves keratin
o Non-specific & produces small fragments
o Optimum temps – 50-55 deg C
o Active in the presence of additives
 SDS, EDTA
o Inactivates DNases & RNases rapidly

Lecture 7 – Sickle Cell/RFLP

 Hb
o T – low affinity, deoxy Hb
o R – high affinity, oxy Hb
o A globin – 16p
o B globin – 11p
 The globin gene
o HbF – fetal – alpha 2 gamma 2
o HbA2 – alpha 2 delta 2 – similar to normal
o HbA – alpha 2 beta 2
o Hb Gower – iota 2 epsilon 2
 Disorders – point mutations
o HbS – AR
 Glu -> Val (A -> T), position 6
o HbC
 Glu -> Lys, position 6
 Detection of beta S
o Adult:
 ASO
 RFLP
 Ddel or MstII restriction endonucleases
o MstII recognizes normal B globin, not mutants
 PCR
o Prenatal:
 FISH
o Preimplantation:
 PCR
 FISH
o DNA sequencing
  Most -ve ------------------------------------------------------------------------> Most +ve
o HbA (bc Glu) HbS (Val) HbC (Lys)
 DNA stains (agarose)
o Ethidium bromide
 Fast and sensitive
 Requires UV
 Mutagenic
o SYBR Safe
 Sensitive
 Expensive
 Needs UV
 Non-mutagenic
o Methylene Blue
 Less sensitive
 Inexpensive
 Reusable
 Electrophoresis performed @ 150V for 30 – 45 mins

Lecture 8 - Bioinformatics

 Nucleotide (DNA) databases: GenBank, EMBL, DDBJ

 Protein databases – UniProt, PIR, Swiss-Prot, ExPASY
 Query sequence
o either amino acid or nucleotide
o min. 15 for BLAST
o can be FASTA, Bare sequence of identifier
 Alignment – regions of greatest statistical similarity between 2 compared sequences
 Score value – measure of quality of alignment; higher the score, better the alignment
 E value – number of different alignments with scores equivalent to or better than
alignment scores expected to occur; lower the E value better the match
 Gene prediction:
o ORF – DNA seq that codes for AAs without any stop codons (UAG, UGA, UAA)
o Each DNA strand can be read in 3 reading frames
o Tool – ORF Finder (NCBI), GENSCAN
 Effective for prokaryotic genes but not eukaryotes bc intron/exon
o Good ORFs:
 Begin with a start codon
 End with an in-frame stop codon
 Many codons close together suggest ORF is not present
 Are of reasonable size (longer is better)
  Long ORF – unlikely – probs a gene
 Features of a gene:
o ORF, start codon (Met), stop codon (TAG, TAA, TGA), terminator seq (prok), TATA
box (euk), Shine Delgarno (prok), Kozak (Euk), Poly A (euk), intron/exon
boundary, CpG islands
 Sequence alignment
o BLAST – Basic Local Alignment Search Tool
 Searches nucleotide or AA database
 BLASTP – protein database using protein query
 BLASTX – protein database, translated nucleotide query
 TBLASTN – translated nucleotide database, protein query
 TBLASTX – translated nucleotide database, translated nucleotide query
 FASTA – DNA query to DNA database, protein query to protein database
 FASTX – translated DNA query to protein database
 TFASTA – protein query to translated DNA database
o FASTA – FAST-ALL
o Input rules
 FASTA
 Single-line description then sequence data
 No blank lines
 Hyphen can represent cap of indiscriminate length
 Bare Sequence
 Lines of sequence data interspersed with numbers +/ spaces
 No blank lines
 Multiple Sequence Alignment Software – used to study phylogenetics
o CLUSTAL W
o COBALT – Constraint based multiple alignment tool
 MASCOT Search Engine – identify, characterize & quantify proteins
o Peptide fingerprint, sequence query & MS/MS ion search
o Any nucleic or AA database in FASTA format can be used