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RADIOBIOLOGY FOR RESIDENTS 2008 Yilun Liu, Ph.D. Molecular Biology Techniques Lecture 1: Nucleic Acids - DNA Fingerprinting: forensic science, paternity/materity test, genetic testing for inherited disease

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slide1

RADIOBIOLOGY FOR RESIDENTS 2008

Yilun Liu, Ph.D.

Molecular Biology Techniques

Lecture 1: Nucleic Acids

- DNA Fingerprinting: forensic science, paternity/materity

test, genetic testing for inherited disease

- gene expression profiling/mutation analysis for identification of disease associated genetic alteration

Lecture 2: Protein

- from protein biochemistry to drug development

slide2

Molecular Biology Techniques: Lecture 1 - Nucleic Acids

Introduction to DNA

(1) chemical structure

(2) chromsome structure

(3) genetic information - species/individual diversity

(4) transcription/translation - from DNA -> RNA -> protein

B. DNA Fingerprinting/Gene Expression Profiling

(1) basis/rationale

(2) Methods

- Restriction Endonuclease

- Agarose Gel Electrophoresis

- Southern Blotting

- Polymerase Chain Reaction (PCR)

- DNA Sequencing

- Northern Blotting

- Microarray

slide3

DNA

  • Deoxyribonucleic acid
  • composed of a chain of nucleotides
  • Each nucleotide contains:
  • (1) phosphate group
  • (2) five carbon sugar (blue)
  • (3) flat aromatic ring (base, orange)
  • There are 4 bases:
      • adenine (A),
  • thymine (T),
  • cytosine (C),
  • guanine (G)
  • A & G are purine (double-ringed)
  • T & C are pyrimidine (single-ringed)

http://www.nvo.com/jin/nss-folder/scrapbookcell/4%20nucleotide.jpg

slide4

DNA

  • DNA = nuleotide chain
  • linked by phosphodiester bond
  • between 5’ phosphate group
  • of one nucleotide to 3’ hydroxyl
  • group of the adjacent nucleotide
  • in cells, DNA exist as
  • double stranded helix.
  • Watson-Crick Base Pairing:
  • A - T
  • G - C

http://www.mcat45.com/images/Nucleotide-bases-mcat.png

slide5

Why packing?

-nucleus ~6um (6x10-6m)

in diameter

-total length of DNA in human

genome ~1.8m (6 ft long!)

slide6

DNA stores genetic information essential for

the development of an organism

“Central Dogma” (Francis Crick, 1956):

transcription

protein

DNA

RNA

translation

replication

slide7

DNA stores genetic information essential for

the development of an organism

Phylogenetic tree

slide9

Homer DNA = Marge DNA

Genetic Polymorphisms

slide10

DNA polymorphism

  • “alternative form of chromosome content”
  • Coding region:
  • (1) nucleotide sequence difference without changing
  • amino acid sequence
slide12

DNA polymorphism

  • “alternative form of chromosome content”
  • Coding region:
  • (1) difference in the nucleotide sequence without
  • changing the amino acid sequence
  • (2) difference in the nucleotide sequence with a
  • change in the amino acid sequence
  • (Genetic polymorphism could lead to disease
  • predisposition)
  • Noncoding region:
    • (1) difference in the nucleotide sequence
    • (2) difference in number of nucleotide repeat units
slide13

VNTR = Variable Number Tandem Repeats

  • small nucleotide repeats
  • ~20-100 base pairs per
  • repeat
  • number of repeats varies
  • among unrelated individuals
slide14

Forensic DNA analysis/DNA Fingerprinting/DNA Profiling

  • - finding the criminal by
  • placing a suspect at a
  • crime scene
  • paternity/maternity test
  • genetic testing for
  • inherited disease
slide15

DNA Fingerprinting Methods

RFLP Analysis

PCR Analysis

slide16

RFLP Analysis

= Restriction Fragment Length Polymophism

Techniques involved:

Restriction Enzyme Digestion

Agarose Gel Electrophoresis

Southern hybridization

slide17

Digestion of DNA

Restriction enzyme = DNA nuclease that cuts double stranded DNA

at a specific sequence (4-10bp)

slide19

Restriction Mapping

  • - a piece of DNA may have multiple cutting sites by a
  • particular restriction enzyme
  • by analyzing the sizes of the digested DNA fragments,
  • one can create a restriction map
  • useful to confirm/identify the identity of a piece of DNA
agarose gel electrophoresis

Agarose Gel Electrophoresis

How to analyze DNA restriction fragments?

slide21

Principles of Electrophoresis

DNA is highly negatively charged

slide23

DNA polymorphism may result in different restriction digest

patterns among different individuals at certain gene locus:

Individual 1

Individual 2

slide25

DNA profiling for inherited disease

visualization of the

digested genomic DNA

on agarose gel

- Too many bands!

slide29

DNA Fingerprinting Methods

RFLP Analysis

Polymerase Chiain Reaction (PCR) Analysis

Advantage: requires much less DNA materials

slide30

PCR developed by Dr. Kary Banks Mullis in 1983

  • a technique to amplify a piece of DNA exponentially in test tube
  • required a heat stable DNA polymerase (Taq) and two oligo primers

25 cycle -> up to 1.7x107 amplification

slide32

DNA profiling for criminal investigation

1 2 3 4 5 6

DNA obtained from

the crime scene

DNA obtained from 6 individual suspects

http://en.wikipedia.org/wiki/Image:D1S80Demo.gif

slide34

Mutation analysis

If you know what gene to analyze…

If you have no idea what gene

to go after….

PCR amplified your gene of interest

from genomic DNA isolated from

normal individual and disease carrier

Comparative Genome

Hybridization

(detect genome wide gain/loss of

DNA content)

DNA sequencing

single-stranded

conformation polymorphism

slide35

DNA sequencing

  • Maxim-Gilbert method
  • (old method, very toxic)
  • -dideoxynucleotide method
slide37

Dideoxynucleotide

sequencing

DNA synthesis requires 3’ OH group

slide38

Single-stranded conformation polymorphism (SSCP)

  • single-stranded DNA forms
  • secondary structure
  • secondary structure may be
  • altered by a single base
  • substitution
  • -mutation(s) can be detected at
  • 50-100% rate within <200bp
  • PCR fragment

Gasser et al., 2007

slide39

Comparative Genome Hybridization

array based

metaphase chromosomes

slide40

What is the consequence of the mutation?

  • (1) Gene expression
  • mutation at promoter
  • mutation at splice site?

Transcription and Splicing

DNA

Exon 3

Exon 1

Exon 2

intron

intron

intron

intron

hRNA

mRNA

to analyze rna transcript northern blot
To analyze RNA transcript- Northern Blot
  • RNA, rather than DNA is blotted
  • Same techniques as Southern blot
  • Can determine levels of RNA expression in tissues, cell types etc.
  • Allows detection of any abnormal size of the transcript
slide42

Northern Blot

occawlonline.pearsoned.com/bookbind/pubbooks/bc_mcampbell_genomics_1/medialib/method/Northernblot.html

slide43

Microarray - global gene expression profiling

Each spot on the array chip

represents one gene

slide45

To confirm or reproduce a disease-associated phenotype

due to the loss of expression of the gene of interest:

Gene knockout

(2) RNA interference

slide46

Gene Knockout

http://www.blackwellpublishing.com/korfgenetics/jpg/300_96dpi/Fig4-16.jpg

slide47

RNA interference

Dicer = RNAse III

RISC = RNA - inducing

silencing complex

slide48

Protein

“Central Dogma” (Francis Crick, 1956):

transcription

protein

DNA

RNA

translation

replication

slide49

Protein Synthesis Overview

Catalyzed by Ribosome

tRNA Met initiates at P site

Correct tRNA binds at A site

slide51

Step 1: decide what host cells to overproduce your protein

  • of interest
  • E. coli
  • Yeast
  • Insect cells
  • Mammalian cells
  • In vitro transcription and translation using wheat germ
  • extract
slide52

Step 2: cloning of bacterial expression plasmid

A typical bacterial plasmid contains:

an origin for replication (ORI)

Drug resistance gene (i.e. AmpR)

slide54

Step 3: protein production

E. coli expression system

IPTG induction

target gene

lac promoter

pET-16b

lac

repressor

slide56

Unlike DNA, not all proteins are negatively charged

Unlike DNA, proteins form high ordered structure

slide59

Step 5: protein purification

commonly used chromatography methods:

size exclusion chromatography

Ion exchange chromatography

affinity chromatography

total protein

lysate

purified

protein

slide63

Analysis of the disease associated mutation on

the biochemical activity of the protein

Example:

WT

nuclease

Disease associated

mutant

nuclease

mutation

Overexpress and purified WT and mutant proteins

assay for gain/loss of nuclease activity in vitro

slide64

What is the consequence of the mutation?

  • Transcription
  • (1) promoter?
  • (2) splicing?
  • Alteration of protein sequence
  • (1) change in biochemical activity?
  • (2) change in protein-protein interaction?
  • (3) change in post-translational modification?
slide65

WT

nuclease

Disease associated

mutant

nuclease

mutation

What’s wrong with this mutant?

Possibility 1: loss of nuclease activity

Possibility 2: loss of crucial protein-protein interaction

slide66

Common methods to study protein-protein interaction

  • Recombinant protein pull down

(2) Co-Immunoprecipitation

(3) Yeast two hybrid screen

slide70

WT

nuclease

Disease associated

mutant

nuclease

mutation

What’s wrong with this mutant?

Possibility 1: loss of nuclease activity

Possibility 2: loss of crucial protein-protein interaction

Possibility 3: loss of DNA binding ability/specificity

slide71

Electrophoretic Mobility Shift Assay (EMSA)

-

agarose/polyacrylamide gel

+

Protein-DNA complex normally has slower mobility due to its larger size than

Unbound/free DNA

slide72

Chomatin IP (ChIP) on chips

Does the mutant protein have the same DNA binding profile

In tumor cells comparing to the WT protein in normal cells?

slide73

protein as drug target

Example:

ICRF-193 =

topoisomerase II inhibitor

(bind and inhibit N-terminal

ATPase domain)