Basic molecular biology and genetic engineering. The central dogma RNA Protein DNA.
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Transcript of Basic molecular biology and genetic engineering. The central dogma RNA Protein DNA.
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
RNA
Protein
DNA
•Proposed by Francis Crick in 1958 to describe the flow of information in a cell.
•Information stored in DNA is transferred residue-by-residue to RNA which in turn transfers the information residue-by-residue to protein.
•It has undergone numerous revisions.
The Central Dogma
Transcription
Translation
Replication
RNA
Protein
DNA
duplication of DNA using DNA as the template
synthesis of RNA using DNA as the template
synthesis of proteins using RNA as the template
DNA Adenine (A) Cytosine (C) Guanine (G) Thymine (T)
RNAAdenine (A) Cytosine (C) Guanine (G) Uracil (U)
The building blocks for DNA and RNA
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
Semi-conservative DNA replication
Daughter DNA molecules contain one parental strand and one newly-replicated strand
DNA polymerase
RNA
Protein
DNA
ATGAGTAACGCGTACTCATTGCGC
ATGAGTAACGCGTACTCATTGCGC
ATGAGTAACGCGTACTCATTGCGC
+
AUGAGUAACGCG
Met-Ser-Asn-Ala
transcription replication
(RNA)
(protein)
(DNA)
translation
codon
Residue-by-residue information transfer
Codon: The sequence of 3 nucleotides in DNA/RNA that encodes for a specific amino acid.
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 =?
Transcription
• Making messenger RNA (mRNA) from part of DNA by RNA polymerase
• Needs a promoter region to begin transcription.
RNA
Protein
DNA
Transcription
Transcription factors + RNA polymerase
closed promoter complex
open promoter complex
initiation
elongation
termination
mRNA
Different cells have different gene transcription patternPromoter: A specific DNA sequence for RNA polymerase and transcription factors binding Different transcription factors recognizes different promoters
Where to start Transcription?
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)
Exon and intron
Prote
in co
ding
Exon
DNA
Prom
oter
+1-tra
nscr
iption
Start Tra
nslat
ion
Start Tra
nslat
ion
Stop
Trans
cript
ion
Stop
Non -
prot
ein co
ding
Intro
n
Annotation of eukaryotic genomes
transcription
RNA processing
translation
AAAAAAA
Genomic DNA
Unprocessed RNA
Mature mRNA
Nascent polypeptide folding
Reactant A Product BFunction
Active enzyme
Gm3
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
Biologic Roles of RNA
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
• 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
What do we actually see?
Hybridization
• Single-stranded DNA or RNA will naturally bind to complementary strands.
ATGAGTAACGCGTACTCATTGCGC
ATGAGTAACGCGUACUCAUUGCGC
DNA
RNA
Probe hybridizationProbe: a single-stranded DNA to detect the presence of a complementary sequence among a mixture of other singled-stranded DNA or RNA
DNA Microarrays
Labeled DNA/RNA mixture flushed over array of short DNA fragments
Laser activation of fluorescent labels
DNA Microarray
Affymetrix array
Each spot (~ 100um) indicates a probe
• measuring the amount of mRNA bound to each probe on the array.
OverviewGREEN 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?Is PCR quantitative?
Cycle #
Log
Targ
et D
NA
Theoretical
-A linear increase follows exponential
Reality
-Eventually plateausTaq polymerase has a half-life of 30 min at 95oC
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.
Real-time PCRReal-time PCR
50-0.005 ng of template- FV Leiden primers
the mostthe most
The leastThe least
Extension
5’ 3’
5’3’5’ 3’
5’3’
Extension continued apply excitation wavelength
5’ 3’
5’3’5’
5’
Taq
Taq
3’
5’3’
Taq
Taq5’
5’
Repeat
DNA binding dyes
BD BD
BD BDBD
BD BD BD
BD BD
Annealing
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)
Human gene
E. coli vector
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
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 - bacteriaTransfection – cell culture or virusTransgenesis – 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.
Human Insulin Production by Bacteria
6) join the plasmid and human fragment
and cut with a restriction enzyme
Transgenesis
The stable integration of a foreign DNA into a host genome
• DNA integrated into the genome• Germline transmission
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.
History of Transgenic mouse
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
Transgenic mice
Mouse Transgenesis Methods
Pronuclear microinjection
Lentivral infection
pros
Relatively simple and efficientLong transgenes possible
Potentially all species
Very efficientSingle copy insertions
No technical equipmentWorks in many species
cons
Random integrationMulticopy insertions
Strain limitationslabor intensive, time consuming
High embryo mortalityExpression level
9.5 kb packaging limitSafety issues (?)
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.