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Basic molecular biology and
genetic engineering
The central dogma
DNA
RNA
Protein
The big three molecules for genetics
• DNA (DexoyribioNucelic Acid)
– Heredity genetic information of an individual
– Encode protein sequences (“genetic code”)
• RNA (RiboNucleic Acid)
– Transfer short pieces of information to cytoplasm
– Provide templates to synthesize protein
• Protein
–
–
–
–
Produced via “translation” of messenger RNA (mRNA)
Each protein has one or more specific functions
Form body’s major components
Carbohydrate and lipid metabolism mediated by proteins
Related terminology
• Genome
– an organism’s complete set of DNA.
• Gene
– basic physical and functional units of heredity.
– specific sequences of DNA encode instructions on how to
make proteins.
• Chromosome
– an organized structure of DNA and protein in the nucleus.
– contains many genes and regulatory elements
• Gene expression
– protein product is being made via transcription and
translation
The central dogma
•Proposed by Francis Crick in 1958 to describe the
flow of information in a cell.
DNA
•Information stored in DNA is transferred residue-byresidue to RNA which in turn transfers the information
residue-by-residue to protein.
RNA
Protein
•It has undergone numerous revisions.
The Central Dogma
Replication
duplication of DNA using DNA as the template
DNA
Transcription
synthesis of RNA using DNA as the template
RNA
Translation
synthesis of proteins using RNA as the template
Protein
DNA & RNA Chains
The building blocks for DNA and RNA
DNA
Adenine (A)
Cytosine (C)
Guanine (G)
Thymine (T)
RNA
Adenine (A)
Cytosine (C)
Guanine (G)
Uracil (U)
DNA: The Basis of Life
• Deoxyribonucleic Acid (DNA)
– Double stranded with complementary strands A-T, C-G
• DNA is a polymer
– Sugar-Phosphate-Base
– Bases held together by H bonding to the opposite strand
Chargaff’s rule
A=T(U) and G=C
Semi-conservative DNA replication
Daughter DNA molecules contain one parental strand and
one newly-replicated strand
DNA polymerase
Residue-by-residue information transfer
(DNA) ATGAGTAACGCG
TACTCATTGCGC
DNA
transcription
(RNA)
RNA
AUGAGUAACGCG
codon
translation
(protein) Met-Ser-Asn-Ala
Protein
replication
ATGAGTAACGCG
TACTCATTGCGC
+
ATGAGTAACGCG
TACTCATTGCGC
Codon: The sequence of 3 nucleotides in
DNA/RNA that encodes for a specific amino acid.
Human genome are completely sequenced at 2003.
What exactly does that mean?
Overview of organizations of life
•
•
•
•
•
Nucleus = bookshelf
Chromosomes = books
Genes = words
Nucleic acid = letters
We are still learning the meaning of the words
– apple =
- zxuriguhwefrhqjrnfg =?
– Zombie =
- manzano =?
We know only small part of protein function
Any genome
Similar
Known
Unknown
Transcription
DNA
RNA
Protein
• Making messenger RNA
(mRNA) from part of DNA by
RNA polymerase
• Needs a promoter region to
begin transcription.
Transcription
closed promoter complex
Transcription factors + RNA polymerase
open promoter complex
initiation
elongation
termination
mRNA
Where to start Transcription?
Different cells have different
gene transcription pattern
Promoter: A specific DNA
sequence for RNA polymerase
and transcription factors
binding
Different transcription
factors recognizes different
promoters
Exon and intron
EXON: In protein coding genes, the segment of a gene that
consists of a sequence of nucleotides that will be eventually
translated into protein.
INTRON: Non coding region of eukaryotic gene
(transcribed into RNA than spliced)
DNA
Annotation of eukaryotic genomes
Genomic
DNA
transcription
Unprocessed RNA
RNA processing
Mature mRNA
Gm3
AAAAAAA
translation
Nascent
polypeptide
folding
Active enzyme
Function
Reactant A
Product B
Translation
tRNA
protein
mRNA
Ribosome
Biologic Roles of RNA
Messenger RNA (mRNA) carries information from DNA to the
ribosomes, and is used as template to synthesize protein.
Transfer RNA (tRNA) is a small RNA that transfers a specific
amino acid to a growing polypeptide chain at the ribosomal site
according to the sequence of a bound mRNA.
Ribosome synthesizes polypeptides under the direction of
mRNA
Mechanism of RNA interference (RNAi):
post-transcriptional gene silencing
shRNA, dsRNA
What is RNA interference (RNAi)?
• RNAi – ability of dsRNA to
silence gene expression in a
sequence-specific manner
• Induced by short interfering
(si)RNAs
• Mechanism: siRNA-directed
cleavage of mRNA via RISC
complex
Analyzing and manipulating a gene or genome
DNA or RNA blotting
What do we actually see?
• Fluorescent chemistries-measurement by either:
– Non-specific (SYBR green/EtBr)
• Pros: inexpensive, no probe design
• Cons: it reports ALL dsDNA formation not just the amplicon
– Specific (probe)
• Pros: increases specificity of the reaction, multiplex and mutation
detection options
• Cons: A bit more expensive, takes time to design probes, need a
unique probe for each target
Hybridization
• Single-stranded DNA or RNA will
naturally bind to complementary
strands.
DNA ATGAGTAACGCG
TACTCATTGCGC
RNA ATGAGTAACGCG
UACUCAUUGCGC
Probe hybridization
Probe: a single-stranded DNA to detect the presence of a
complementary sequence among a mixture of other singledstranded DNA or RNA
DNA Microarrays
Labeled DNA/RNA mixture flushed
over array of short DNA fragments
Laser activation of fluorescent labels
DNA Microarray
• measuring the amount of mRNA bound to each probe on the
array.
Affymetrix array
Each spot (~ 100um) indicates a probe
Overview
GREEN represents Control DNA
RED represents Sample DNA
YELLOW represents a combination of
Control and Sample DNA
BLACK represents areas where
neither the Control nor Sample DNA
.
Polymerase Chain Reaction
• Goal: to amplify a low level DNA template for further
analysis or manipulation
• Need :
– Primer: A set of single stranded DNA hybridize to the both
end of target region
– Taq polymerase: a thermostable DNA polymerase
– Template DNA
Is PCR quantitative?
-A linear increase follows exponential
-Eventually plateaus
Taq polymerase has a half-life of 30 min at 95oC
Log Target DNA
Theoretical
Reality
Cycle #
Real-time PCR
Through the use of fluorescent molecules, real-time PCR
allows us to ‘see’ the exponential phase so we can calculate
how much we started with.
50-0.005 ng of template- FV Leiden primers
The least
the most
DNA binding dyes
5’
3’
Annealing
5’
3’
3’
BD
5’
Taq
3’
BD
BD
BD
BD
Taq
Taq
BD
BD
5’
Extension
BD
3’
BD
BD
Repeat
Taq
5’
3’
Extension continued apply
excitation wavelength
Fluorescence detection
Emission
Filter
Excitation
Filter
Light Source
Detector
What does “cloning” mean?
Clone: a collection of molecules, cells or individuals, all identical
to an original one
•
•
•
•
To "clone a gene" is to make many copies of it
Gene can be an exact copy of a natural gene
Gene can be an altered version of a natural gene
Recombinant DNA technology makes it possible
Recombinant DNA
• taking a piece of one DNA, and combining it with another strand of
DNA that would not normally occur together.
• In order to do so, we need to • Copy it (PCR)
• Cut it (restriction enzyme digestion)
• Paste it (ligation)
E. coli vector
Human gene
Restriction Enzymes
– They cut the DNA at specific sequence.
– Different restriction enzymes have different recognition
sequences.
Pasting DNA
• Two pieces of DNA can be
fused together by adding
DNA ligase
– Hybridization –
complementary base-pairing
– Ligation – fixing bonds with
single strands
Recombinant DNA Techniques
Genetic Engineering
• To transport a specific segment of DNA from one
organism to another
Put human gene to E.coli
Introducing foreign into organism
• The process whereby a DNA sequences are introduced by
biologial, biochemical or physical processes.
Transformation - bacteria
Transfection – cell culture or virus
Transgenesis – animal
• Biological: virus infection
• Biochemical: DEAE-dextran, calcium phosphate, and
liposome-mediated transfection methods.
• Physical: direct micro-injection of materials, biolistic particle
delivery and electroporation.
The Power of Recombinant DNA Technology –
Human Insulin Production by Bacteria
Human Insulin Production by Bacteria
and cut with a restriction enzyme
6) join the plasmid and human fragment
Human Insulin Production by Bacteria
Mix the recombinant plasmid with
bacteria.
Human Insulin Production by Bacteria
fermentor
Transgenesis
The stable integration of a foreign DNA into a host genome
• DNA integrated into the genome
• Germline transmission
History of Transgenic mouse
1980-81:Several groups (Gordon et al., Brinster, Constantini et al,
Lacy et al.) reported the development of transgenic mice by
microinjecting genes into the pronucleus of a fertilized egg.
1982: The first visible phenotype was shown by Dr. R.Palmiter and
colleagues in mice overexpressing rat growth hormone.
A photograph of a giant transgenic mouse was on the cover of Nature.
Transgenic mice
Foreign DNA introduced into the germ line of mice by
pronuclear injection; random gene insertion
- specific genes to study pathogenesis of the disease
- gene expressed using viral promoters/cell specific
promoter
Promoter
gene
pA
Mouse Transgenesis Methods
pros
cons
Relatively simple and efficient
Long transgenes possible
Potentially all species
Random integration
Multicopy insertions
Strain limitations
labor intensive, time consuming
Very efficient
Single copy insertions
No technical equipment
Works in many species
High embryo mortality
Expression level
9.5 kb packaging limit
Safety issues (?)
Pronuclear microinjection
Lentivral infection
Transgene DNA
founder
What is a Knockout Mouse?
• A mouse in which a very specific endogenous gene
has been altered in such a way that interferes with
normal expression, i.e. it has been knocked out.
Gene knockout overview
Analysis in human