Molecular Biology Primer

1
Molecular Biology Primer

• Lecture 3 September 8, 2005
• Algorithms in Biosequence Analysis
• Nathan Edwards - Fall, 2005

2
Biotechnology

• Use the machinery of molecular biology to study
• DNA
• Genes
• Transcription
• Splicing
• Proteins
• Polymorphism
• Coupled with samples to compare, we have systems
  biology.

3
Biotechnology

• Use (abuse) specific proteins from biology to
  manipulate the sample
• Biochemistry techniques make the result
  observable
• Observation is digitized and (often) processed
  for consumption

4
DNA Manipulation

• Restriction enzymes
• Cut DNA into smaller pieces
• Hybridization
• Join two complementary single stranded DNA
  molecules
• Polymerase Chain Reaction (PCR)
• Make lots of copies of a small DNA region
• Cloning / Vectors
• Make lots of copies of a large DNA region

5
Observation

• Electrophoresis
• Separate based on size and shape
• Fluorescence
• Brightness / Color
• Arrays
• Compact / Position determined

6
Restriction Enzymes

• Cut DNA at specific sequence motifs
• EcoRI
• EcoRII
• EcoNI
• IUPAC Symbols represent multiple possible bases

GAATT C C TTAAG
CCWGG GGWCC
CCTNNN NNAGG GGANN NNNTCC
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IUPAC Amiguity Codes

• International Union of Pure and Applied Chemistry

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DNA Denaturation Hybridization
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DNA Denaturation Hybridization

• DNA will hybridize with any single DNA strand
  with the correct (complementary) DNA sequence
• Short oligonucleotides can hybridize to the DNA
  strand instead!

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Polymerase Chain Reaction
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PCR
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Cloning

• Result is a colony of bacteria that all contain
  the inserted sequence.
• The inserted sequence can later be extracted
  using similar techniques
• Suitable for larger pieces than PCR

13
Electrophoresis
14
Florescence / Dyes

• Add colors for optical detection

15
DNA Sequencing

• Similar to PCR, but only 1 primer
• A few special, labeled nucleotides terminate
  extension
• Get a random mix of different length fragments
  color-coded by final nucleotide

16
DNA Sequencing

• Electrophoresis separates the fragments by size

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DNA sequencing
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DNA Sequencing
19
Genotypes

• Everybodys genome is essentially the same
• but the differences matter
• We each have two copies!
• Genomic variation
• may have a direct or indirect effect
• records heredity
• provides genomic landmarks

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Genotyping Technology
Probes for each allele
Genomic DNA
SNP site
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Genotyping Technology
Genomic DNA
SNP site
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Genotyping Technology
Genomic DNA
SNP site
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mRNA / Transcription

• Reverse transcriptase
• Makes a DNA copy of the mature RNA
• After introns are spliced out.
• cDNA complementary DNA
• cDNA can then be amplified by PCR, and sequenced
• All of the previous techniques can now be applied
  to the cDNA
• Sequencing ESTs
• Quantitation microarrays

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Gene Expression

• Quantify the amount of gene expression
• Measure mRNA
• cDNA hybridization and fluorescence
• Many genes at once
• Usually compares two samples
• Treated, control
• Tumor, non-tumor
• Time course
• Lots of images from Terry Speeds website

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Gene Expression

• Label samples

• Hybridize and scan

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Gene Expression Data
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Proteins

• Digestion enzymes
• Cuts proteins into pieces
• Separation by physical or chemical properties
• water affinity, mass, AA composition
• Cant amplify proteins!
• Have to enrich the existing sample
• Mass Spectrometry
• Antibodies

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2D Gel-Electrophoresis

• Protein separation
• Molecular weight (Mw)
• Isoelectric point (pI)
• Staining
• Birds-eye view of protein abundance

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2D Gel-Electrophoresis
Bécamel et al., Biol. Proced. Online 2002494-104
.
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Mass Spectrometer

• ElectronMultiplier(EM)

• Time-Of-Flight (TOF)
• Quadrapole
• Ion-Trap

• MALDI
• Electro-SprayIonization (ESI)

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Mass Spectrometer (MALDI-TOF)
UV (337 nm)
Microchannel plate detector
Field-free drift zone
Source
Pulse voltage
Analyte/matrix
Ed 0
Length D
Length s
Backing plate (grounded)
Extraction grid (source voltage -Vs)
Detector grid -Vs
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Mass Spectrum
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Mass is fundamental
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Mass Spectrum
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Mass Spectrum

• Isotope Cluster
• 12C 99
• 13C 1

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Peptide Mass Fingerprint
Cut out 2D-GelSpot
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Peptide Mass Fingerprint
Trypsin Digest
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Peptide Mass Fingerprint

• Trypsin digestion enzyme
• Highly specific
• Cuts after K R except if followed by P
• Protein sequence from sequence database
• In silico digest
• Mass computation
• For each protein sequence in turn
• Compare computer generated masses with observed
  spectrum

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Protein Sequence

• Myoglobin - Plains zebraGLSDGEWQQV LNVWGKVEAD
  IAGHGQEVLI RLFTGHPETL EKFDKFKHLK TEAEMKASED
  LKKHGTVVLT ALGGILKKKG HHEAELKPLA QSHATKHKIP
  IKYLEFISDA IIHVLHSKHP GDFGADAQGA MTKALELFRN
  DIAAKYKELG FQG

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Protein Sequence

• Myoglobin - Plains zebraGLSDGEWQQV LNVWGKVEAD
  IAGHGQEVLI RLFTGHPETL EKFDKFKHLK TEAEMKASED
  LKKHGTVVLT ALGGILKKKG HHEAELKPLA QSHATKHKIP
  IKYLEFISDA IIHVLHSKHP GDFGADAQGA MTKALELFRN
  DIAAKYKELG FQG

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Peptide Masses

• 1811.90 GLSDGEWQQVLNVWGK
• 1606.85 VEADIAGHGQEVLIR
• 1271.66 LFTGHPETLEK
• 1378.83 HGTVVLTALGGILK
• 1982.05 KGHHEAELKPLAQSHATK
• 1853.95 GHHEAELKPLAQSHATK
• 1884.01 YLEFISDAIIHVLHSK
• 1502.66 HPGDFGADAQGAMTK
• 748.43 ALELFR

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Peptide Mass Fingerprint
YLEFISDAIIHVLHSK
GHHEAELKPLAQSHATK
GLSDGEWQQVLNVWGK
HPGDFGADAQGAMTK
VEADIAGHGQEVLIR
HGTVVLTALGGILK
KGHHEAELKPLAQSHATK
ALELFR
LFTGHPETLEK
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Stable Isotope Labeling
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Stable Isotope Labeling

• SILAC Lysine with 12C6 vs 13C6

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Tandem Mass Spectrometry(MS/MS)
Precursor selection
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Tandem Mass Spectrometry(MS/MS)
Precursor selection collision induced
dissociation (CID)
MS/MS
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Peptide Fragmentation
yn-i-1
-HN-CH-CO-NH-CH-CO-NH-
CH-R
Ri
i1
R
i1
bi1
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Peptide Fragmentation
Peptide S-G-F-L-E-E-D-E-L-K
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De Novo Interpretation
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De Novo Interpretation
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Bioinformatics Problems

• Inverse problems
• Identify or quantify the DNA, RNA, Protein from
  observations
• Incorporation of EST / peptide evidence into gene
  finders
• Genome assembly
• Disease association
• Hybridization assay design