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 & RNA Chains

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

Chargaff’s ruleA=T(U) and G=C

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.

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 =?

Unknown

Similar

Known

Any genome

We know only small part of protein function

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

Translation

Ribosome

tRNA

mRNA

protein

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

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

• 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

Detector

Fluorescence detectionFluorescence detection

EmissionFilter

Light Source

ExcitationFilter

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

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 - 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.

The Power of Recombinant DNA Technology – Human Insulin Production by Bacteria

Human Insulin Production by Bacteria

6) join the plasmid and human fragment

and cut with a restriction enzyme

Mix the recombinant plasmid with bacteria.

Human Insulin Production by Bacteria

fermentor

Human Insulin Production by Bacteria

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 (?)

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