Basic Laboratory Techniques

1
Basic Laboratory Techniques

• Microarray Database Systems
• 9/23/2002

2
Outline

• Basic genetic engineering tools
• labeling, gel electrophoresis, hybridization
• DNA sequencing
• DNA cutting
• DNA amplification
• DNA cloning
• PCR

3
Why Details?

• to study papers
• to understand and communicate with biologists
• to record important procedures that affect the
  data in databases or analysis tools
• to integrate data coming from different domains

4
Isolation of DNA and RNA

• open cells in the sample
• cell disruption remove cell wall, membrane
• as gentle as possible
• separation of nucleic acids from others
• deproteinisation
• purification recovery of nucleic acids
• DNA RNase to digest RNA
• RNA oligo(dT)-cellulose to bind poly(A) tails of
  eukaryotic mRNAs

5
Syntheis of cDNA
mRNA
AAAAAA 3

• prepare mRNA
• 3 poly(A) tail
• insert a primer
• in case of full length synthesis, oligo(dT)
  matching 3-end poly(A) tail of the mRNA
• reverse transcription
• all four dNTPs supplied (can be labeled)
• DNA polymerases

AAAAAA 3
HO - TTTTTT 5
AAAAAA 3
TTTTTT 5
cDNA
TTTTTT 5
OH
TTTTTT 5
double-strand cDNA
6
Nucleic Acid Labeling

• End Labeling
• adding a modified, or radioactive, nucleotide to
  one end of a DNA fragment
• 5-End labeling add a radioactive phosphate
• 3-End labeling add one or more nucleotides

7
Nucleic Acid Labeling

• By Primer Extension
• denature DNA
• anneal with designed primers
• synthesize DNA
• DNA polymerase
• incorporating labeled nucleic acids

5
3
5
3
HO
Primer
5
3
Primer
DNA polymerase I
8
Non-radioactive Labeling

• safer, faster
• fluorescent or non-fluorescent

9
Gel Electrophoresis

• label molecules
• with radioactive isotopes or fluorescent dyes
• charge the molecules and put them in one end of a
  gel medium with strong electric field
• the molecules move in the gel with speed
  inversely proportional to their size
• logarithmic relationship
• measure the relative weight through the relative
  offset

10
Genome Sequencing

• Chromosome
• a long strand of DNA (molecule)
• Locus (loci)
• the location of a gene in a chromosome
• Physical Map
• the exact location, in base pairs, of a gene in
  a chromosome

11
Sequencing Top-down

• Prepare 4 groups of DNAs
• Break the DNAs into pieces end with A, T, G, C,
  respectively
• Maxam-Gilbert (chemical)
• Sanger-Coulson (enzymatic)
• Run the 4 DNA groups using gel electrophoresis in
  4 lines
• Read exact sequence
• up to 700 bps long

12
Automated DNA Sequencing

• four different, fluorescent dye-labeled chain
  terminators
• one tube
• one gel lane
• read by laser
• automated sample processing
• high throughput

13
Sequencing Shotgun Method

• Break DNAs into pieces randomly
• Select a subset for further processing
• amplification
• Sequence the pieces
• Paste the pieces together
• computer

14
DNA Cutting

• Restriction Enzyme
• Proteins that cut DNA at specific points called
  restriction sites
• determined by the local base sequences
• may leaving sticky ends
• Example EcoRI looks for GAATTC
• GAATTC gt G AATTC
• CCAGAATTCTCG gt CCA GAATTCTCG
• GGTCTTAAGAGC GGTCTTAA GAGC

15
DNA Cloning

• Generate an initial DNA fragment
• Join to a vector (DNA cutting involved)
• Introduce into a host cell and let the cell
  multiply
• extract the inserts
• recombinant refers to DNA produced this way

16
Popular Vectors

• Plasmids
• circular DNA exists in bacteria
• afford up to 15 kbp inserts
• Phages
• viruses that infects bacteria
• for inserts up to 25 kbp (limited by capsule)
• YAC (yeast artificial chromosome)
• extra, artificially made chromosome to yeast
• for large inserts (a million bps)

17
Libraries

• Library A collection of independent clones
• Genomic Library a set of clones representing the
  entire genome of an organism
• represent engire genome as a set of overlapping
  cloned fragments
• randomly
• maintained in stable form with no
  misrepresentation of sequences
• cDNA Library
• from mRNA

18
Polymerase Chain Reaction

• Iterations of phases
• denature double-strand DNA
• heat
• convert single strands into double strands
• need primer
• Taq thermostable polymerase

5
3
5
3
5
3
primer
primer
3
5
Taq
5
3
primer
primer
3
5
Taq
8
16
19
Probe and Target

• Probes
• oligonucleotides or clones (DNA, RNA) with known
  sequences
• Targets
• biomaterials under study

20
Hybridization

• Joining together (artificially) separated nucleic
  acid strands
• denature probes and/or targets
• into single strands, by heat (90 oC)
• either one can be immobilized
• anneal into double strand
• 65 68 oC, in plastic bag or chamber

21
Southern (Northern) Blotting

• Run targets DNA (RNA) in gel
• Denature DNA into single strands (Northern)
• Transfer from gel into membrane
• immobilize targets
• Hybridization with labeled probes
• Wash out non-hybridized materials
• Read out image data

22
Microarray

• immobilize massive probes on a chip
• hybridize with labeled target samples
• extract relative abundance of the base pairing
• investigate gene expression

Labeled sample
Hybridization
Scanning
23
Impact of Microarray

• General
• massive parallel from gene to genome study
• functional genomics more than sequence
• Technical
• advanced image analysis, noise handling
• large-scale data storage and management
• large-scale data processing
• heterogeneity in technology and data format
• Long-term
• full feedback loop from experiment to analysis