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Genetic Engineering
This is a mind map about genetic engineering, with a detailed introduction and comprehensive description. I hope it can be helpful to interested friends.
Edited at 2023-11-24 01:34:49- cáncer de pulmón
El cáncer de pulmón es un tumor maligno que se origina en la mucosa bronquial o las glándulas de los pulmones. Es uno de los tumores malignos con mayor morbilidad y mortalidad y mayor amenaza para la salud y la vida humana.
- diabetes
La diabetes es una enfermedad crónica con hiperglucemia como signo principal. Es causada principalmente por una disminución en la secreción de insulina causada por una disfunción de las células de los islotes pancreáticos, o porque el cuerpo es insensible a la acción de la insulina (es decir, resistencia a la insulina), o ambas cosas. la glucosa en la sangre es ineficaz para ser utilizada y almacenada.
- sistema digestivo
El sistema digestivo es uno de los nueve sistemas principales del cuerpo humano y es el principal responsable de la ingesta, digestión, absorción y excreción de los alimentos. Consta de dos partes principales: el tracto digestivo y las glándulas digestivas.
Genetic Engineering
- cáncer de pulmón
El cáncer de pulmón es un tumor maligno que se origina en la mucosa bronquial o las glándulas de los pulmones. Es uno de los tumores malignos con mayor morbilidad y mortalidad y mayor amenaza para la salud y la vida humana.
- diabetes
La diabetes es una enfermedad crónica con hiperglucemia como signo principal. Es causada principalmente por una disminución en la secreción de insulina causada por una disfunción de las células de los islotes pancreáticos, o porque el cuerpo es insensible a la acción de la insulina (es decir, resistencia a la insulina), o ambas cosas. la glucosa en la sangre es ineficaz para ser utilizada y almacenada.
- sistema digestivo
El sistema digestivo es uno de los nueve sistemas principales del cuerpo humano y es el principal responsable de la ingesta, digestión, absorción y excreción de los alimentos. Consta de dos partes principales: el tracto digestivo y las glándulas digestivas.
- Recommended to you
- Outline
Genetic Engineering
Gene
concept
A specific nucleotide sequence that has a genetic effect on the DNA molecule
Guide the synthesis of important substances such as proteins in the human body and maintain the normal physiological functions of the human body.
Material level: a deoxyribonucleic acid (DNA) sequence on a chromosome.
Functional level: carrier of genetic information, encoding protein and ribonucleic acid (RNA) molecular functions, and regulating gene expression.
nucleic acid
Classification
DNA (DNA)
Nucleus, mitochondria, chloroplasts, plasmids...
carrier of genetic information
RNA (RNA)
Cytoplasm, nucleus
Classification
Ribosomal RNA (rRNA)
components of ribosomes
Transfer RNA (tRNA)
transport amino acids
Messenger RNA (mRNA)
Transmit genetic information from DNA and guide protein synthesis
chemical components
Element composition: C, H, O, N, P
Molecular composition:
base
purine base
pyrimidine base
The inner side of the spiral
Pentose
Ribose (U)
Deoxyribose (T)
Phosphoric acid
The skeleton of the spiral chain
Basic unit: nucleotide
A brief history
Mendel: The Pea Experiment
Discover the laws of inheritance: the same pair of factors are separated, and the different pairs of factors are freely combined
Proposed for the first time "genetic factors" (i.e. genes)
Morgan: Drosophila Experiments
genes on chromosomes
Inherited Genetic Chains & Law of Exchange
Watson & Crick: DNA double helix structure model
DNA right-handed double helix structure
DNA replication mechanism: semi-conservative replication
Genetic Engineering
The basis of birth
Three major theoretical discoveries
The carrier of genetic information is DNA, not protein [Pneumococcus pneumoniae transformation experiment]
Double helix structure model of DNA molecule & semi-conservative replication mechanism
Central Dogma & Operator Theory and Decoding of Genetic Code
Theoretical basis
The basic structure of DNA in all organisms is the same. Different genes have the same material basis, so genes can be recombined and interchanged.
Genes can be cut and transferred, ensuring that genetic engineering can operate on genes without affecting their functions.
The genetic code is universal, and there is a correspondence between polypeptides and genes, regardless of species.
Genes can pass genetic information to the next generation through replication.
Three major inventions in technology
In vitro cutting and joining technology of DNA molecules
Use of genetic engineering vectors
Xu Lei analysis of DNA molecules, agar gel electrophoresis, hybridization technology...
1937: The first year of genetic engineering
concept
It refers to the use of enzymatic methods to recombine heterologous genes & carrier DNA in vitro, and the resulting recombinant DNA is introduced into host cells, so that the heterologous genes can be replicated and expressed in the host cells, thereby achieving the transformation of recipient biological species or traits and mass production. Produce biological varieties and products needed by human beings,
Also known as: molecular cloning or recombinant DNA technology.
main content
From the genome of a biological organism, the DNA fragment containing the gene of interest is isolated.
The exogenous DNA fragment carrying the target gene is connected to a self-replicating vector molecule with a selection mark to form a recombinant DNA molecule.
The recombinant DNA molecules are transferred to appropriate recipient cells (host cells) and propagated with them.
From a large number of cell propagation populations, the recipient cells that have obtained the recombinant DNA molecules are screened out, and the target genes that have been amplified are screened out.
The target gene is cloned into an expression vector and introduced into host cells so that it can achieve functional expression under a new genetic background and produce substances needed by humans.
basic process
Five basic operating units: cutting, connecting, transferring, adding, and inspecting
Isolate DNA containing the gene of interest
Restriction enzymes cut DNA fragments (cut)
Plasmid is isolated from E. coli and digested (digested)
Ligase connects the target gene & plasmid for enzyme digestion (connected to: DNA recombination)
Cutting and splicing: in vitro recombination of DNA
Introduce the recombinant plasmid into the host cell (transform)
Clone the target gene and screen for expression (amplification and detection)
Test: Screening and identification of transformants
Transformation and amplification: transformation and amplification of recombinant DNA molecules
Genes of any organism → any other receptor cell that has nothing to do with it
A certain segment of DNA → replicated in the recipient cell → prepare a large number of purified DNA fragments
Four elements
Tool enzyme
Target gene (preparation)
gene vector
gene receptor
DNA
Classification
chromosomal DNA
Obtain the target gene
Plasmid DNA
carrier
Virus, phage DNA
Construct cloning vectors, isolate target genes & gene expression regulatory factors
mitochondrial chloroplast DNA
Obtain the target gene
extract
Preparing biological materials
Rich DNA content with few impurities
Plasmid DNA
Bacterial liquid culture → late logarithmic growth phase
plant DNA
Young parts or seedlings, reduce starch/sugar accumulation → interfere with DNA extraction
animal DNA
Remove areas with high enzyme activity
lysed cells
Proper lysis to avoid DNA strand breaks
Prokaryotes: lysozyme, ultrasound, NaOH, SDS treatment or boiling, freezing treatment
Eukaryotes: Crushed → Same as above
Isolate & purify DNA
Cell fluid after lysis → + protein denaturant [phenol/chloroform/isoamyl alcohol/SDS...] (denaturation of protein) → centrifugation (removal of impurities such as protein) → organic solvent [ethanol/isopropyl alcohol] (aggregation and precipitation of DNA) → Remove solution → Crude DNA → Further purification [phenol/chloroform: extraction, 70% ethanol]
Detection: Gel electrophoresis technology
principle
mobility
Migration speed of electrophoretic molecules in an electric field
Proportional to the electric field strength & its own net charge
Inversely proportional to the friction coefficient of dielectric molecules
The DNA backbone itself is negatively charged and moves toward the positive electrode in the electric field;
Different DNA molecular weights and configurations are different → electrophoretic mobility is different → different zones.
Separate the components of a mixture of proteins or nucleic acid molecules.
type
agar gel electrophoresis
"Small, wide": separation degree: poor; separation range: better than
polyacrylamide gel
The higher the gel concentration, the smaller the pores and the stronger the resolving power.
Tool enzyme
restriction endonuclease
concept
Substrate: circular or linear double-stranded DNA
Recognition: Special nucleotide sequences
Break: (appropriate reaction conditions) specific phosphodiester bonds of each chain
Produce: DNA fragments of 3'-OH and 5'-P genes
endodeoxyribonuclease
type
Type I enzyme
Recognition site is far away, arbitrary cleavage, poor specificity, requires cofactors
Type II enzyme ✓
Recognition site: specific site in/near the defined site, specific cleavage, strong specificity, consistent recognition & cleavage sequence, no need for cofactors or only Mg2
Type III enzyme
Cutting outside the recognition site, the recognition site is an inverted repeat sequence, irregular, and rarely produces completely cut fragments
Naming principles
Composition: strain number, strain name, isolation sequence
The first letter of the genus name is capitalized, the first two letters of the species name are lowercase, and the order of separation (Roman letters)
Mechanism
recognition sequence
Restriction enzymes can recognize special nucleotide sequences on double-stranded DNA.
The same molecule is short and has a high probability of occurrence.
Mode of action
Enzyme cutting characteristics
Recognition length
4~8 pairs of bases
cutting position
Recognize the interior or both sides of the sequence
Identify structure
palindrome
Type of enzyme
Rare Dicer
isoschiase
Recognition sequence is the same
Homogenase
The recognition sequences are different, but the same sticky ends are obtained
Cutting method
Sticky ends (staggered cuts)
5' sticky end (5' short)
3' sticky end (3' short)
Flat ends (cut flush)
reaction system
Reaction substrate (DNA)
Endonuclease dosage (determined by activity)
Reaction buffer (optimum pH7.5)
Appropriate temperature (mostly 37℃)
Enzyme digestion identification: gel electrophoresis technology
DNA ligase
concept
Catalysis: between the adjacent 3'-OH and 5'-P ends of the DNA molecule
Formed: phosphodiester bond
That is: sealing the single-stranded gap between adjacent nucleotides on double-stranded DNA.
There are two main types of
E. coli-DNA ligase
Sticky end, requires cofactor: NAD
The same restriction enzyme or two homogeneous enzymes
T4-DNA ligase
Sticky ends, flat ends, requires cofactor: ATP
The same or two restriction enzymes
Connections with non-sticky ends and non-flat ends
exonuclease
flat ends
terminal nucleotidyl transferase
Modified into complementary sticky ends
alkaline phosphatase
Modify 5'-P to 5'-OH to prevent the carrier from self-cyclization
The main connection methods of DNA
Homologous endonuclease → sticky end
homotailase→sticky end
different sticky ends
Cut flat, fill flat
Artificial sticky ends - 5 protruding' ends
Make up the level
Artificial sticky ends - 3 protruding' ends
Artificial sticky ends - flat ends
Replacement of sticky ends
Other tool enzymes
DNA polymerase
PCR amplification
reverse transcriptase
Construct cDNA library
T4 polynucleotide kinase
S1 nuclease
Preparation of target gene
target gene
concept
☞A specific gene/DNA fragment that has been/is to be isolated, transformed, amplified and expressed, can encode a certain product/trait
Also known as: special gene or target gene
source
Eukaryotic chromosomes genome (human & animal)
prokaryotes chromosomes
plasmid, virus
Mitochondria, chloroplasts...
method
Direct decomposition method
principle
Chromosomal DNA → Restriction endonuclease: Cut → Link all fragments → A certain vector → Transfer into E. coli → Propagate → Screen with appropriate methods → Recombinant colonies containing the target gene → Extract DNA → Enzyme digestion → Obtain the target gene.
object
Prokaryotic genomes are small, genes are easy to locate, or DNA molecules with known nucleotide sequences.
advantage
Quick and easy, product purity is high. target gene
gene library method
principle
All the genetic information of a certain organism's genome → cloning vector → storage: a clone subpopulation of recipient bacteria (the genome library of this organism).
The group of recipient bacteria that contains all the genes in the genome of a certain organism is called: the genome library of the organism
chemical synthesis
When the sequence information is known, it is the most convenient way to obtain the target gene!
PCR amplification method (polymerase chain reaction)
substance
In vitro simulation of in vivo DNA replication
principle
cycle
transsexual
94℃
Heating/alkaline, DNA double strand → hydrogen bond break → single strand
annealing
55℃
Template & primer renaturation
extend
72℃
Specific DNS sequence: geometric exponential growth
PCR reaction process/reaction steps
Denaturation of template DNA
After the template DNA is heated to 94~95°C for a certain period of time, the double-stranded DNA of the template DNA or the double-stranded DNA formed by PCR amplification is dissociated into a single strand so that it can bind to the primer and prepare for the next round of reaction;
Annealing (renaturation) of template DNA and primers
After the template DNA is heated and denatured into a single strand, the temperature drops to about 55°C, and the primer pairs with the complementary sequence of the template DNA single strand;
primer extension
Under the action of Taq DNA polymerase, the DNA template-primer conjugate uses dNTP as the reaction raw material and the target sequence as the template. According to the principles of base pairing and semi-conservative replication, a new semi-conservative replication strand complementary to the template DNA strand is synthesized. .
PCR reaction system
reaction buffer
Substrate: dNTP (4 kinds of deoxynucleotides: dATP, dGTP, dTT, dCTP)
Primers×2
Template: DNA molecule
Taq enzyme (DNA polymerase)
Mg2 (enzyme cofactor)
PCR characteristics
high sensitivity
Strong specificity
Simple, fast and reproducible
Low requirements for specimen purity
carrier
The concept of gene carrier
Tools or carriers that introduce foreign DNA or target genes into appropriate host cells
The carrier should meet the conditions
The relative molecular mass is small and suitable for receiving the target gene;
It can carry out independent and stable self-replication within the host cell, and can still maintain stable replication and genetic characteristics when foreign DNA is inserted.
It can provide selectable markers for host cells (receptors) and have identifiable phenotypic characteristics for easy screening;
There is an appropriate single restriction endonuclease cutting site in its DNA sequence. This site is located in the non-essential region for DNA replication. That is, after being cut by a certain restriction endonuclease, the plasmid DNA can be closed and opened to admit foreign matter. Source DNA fragments without losing your own fragments.
Plasmid
concept
Double helix closed circle DNA molecule
It is independent of chromosomes for replication and inheritance, and relies on host-encoded enzymes and proteins for replication and transcription.
Vs chromosomes
All related to biological genetics
Chemically different
Chromosomes: Protein & DNA
Plasmid: DNA
type
Tight type
There is only one copy, or at most there are only a few copies.
Relaxed
Commonly used plasmid vectors
Escherichia coli plasmid vector
Two antibiotic resistance genes
Lactose operon: lacZ gene → β-galactosidase → hydrolysis X-gal (lacZ blue-white spot screening)
Plasmid DNA extraction
selective extraction
Under milder conditions, lysozyme is used to lyse bacteria, and the resulting cell fragments are separated by high-speed centrifugation due to differences in relative molecular weight between chromosomal DNA and plasmid DNA. Plasmid DNA with a relatively small molecular weight remains in the supernatant, and is precipitated with ethanol to obtain crude plasmid DNA.
Alkaline SDS method
It selectively denatures the double-helix open-stranded DNA in the chromosome within the pH range of 12.0-12.5, while leaving the closed-loop double-stranded DNA unchanged. After neutralization with sodium acetate, SDS causes the protein-SDS complex and DNA with high relative molecular mass to precipitate, and then the plasmid DNA is left in the supernatant and separated by high-speed centrifugation.
Plasmid DNA purification
Alkaline sucrose density gradient centrifugation
Chromosomal double helix open-stranded DNA is easily denatured, while closed-circle double-stranded plasmid DNA is not easily denatured. In the sucrose concentration gradient, plasmid DNA settles faster than denatured DNA and is separated and purified.
Cesium chloride-ethidium bromide (Et-Br) density gradient centrifugation method
The open-stranded DNA of the double helix of the chromosome is easy to combine with the dye Et-Br and the density is reduced. The plasmid DNA is not suitable for binding because it is tightly closed, and the density is high, so it is separated by centrifugation.
Nitrocellulose membrane adsorption method
Nitrocellulose membrane adsorbs single-stranded DNA, and chromosomal DNA is easily denatured to form single-stranded DNA, which is separated from plasmid DNA.
Transform and build
Delete non-essential areas
Add new genetic marker genes
Introduce DNA sequences with multiple restriction enzyme recognition and cleavage sites, that is, multiple cloning sites
lambda phage
Phage
It cannot reproduce independently of host cells and can only grow in living cells & is strictly specific to host cells.
Main types
insert vector
replacement vector
Other carriers
genetic recombination
concept
Target gene & vector: in vitro combination → recombinant.
Reorganization method
Mainly sticky ends
direct bonding
Simple and efficient
Add tail bonding
Using terminal nucleotidyl transferase to create sticky ends at DNA ends
Main tool enzyme: T4-DNA ligase (5'-P and 3'-OH)
DNA recombination
① Ligation of sticky ends produced by the same endonuclease
② Ligation of sticky ends produced by homologase
③Connection of different sticky ends
④Connection of artificial sticky ends (5’ protruding end, 3’ protruding end, flat end)
⑤ Replacement of sticky end
Recombination rate = index of recombination components containing exogenous DNA / number of vector molecules, routine experimental conditions: 25~75%
Gene transformation & expression
Target gene is introduced into recipient cells
Host cell (receptor cell)
concept
Cells receiving foreign gene DNA during transformation, transduction & hybridization
is recombinant amplification place
type
prokaryotic cells
Escherichia coli, Bacillus subtilis
eukaryotic cells
Yeast (mainly)
Features
Unicellular
Non-pathogenic, safe
Competence
Competence
Host cells can absorb exogenous DNA molecules and effectively act as transformation receptors in certain physiological states.
depending on
The physiological state of the receptor cell itself
Configuration & size of recombinant DNA molecules
General host cells in logarithmic growth phase: transformation ability Max
competent cells
Cells in a physiological state that can absorb external DNA molecules.
Convert
Ca2-induced transformation of intact bacterial cells
electroporation transformation
Conversion rate
concept
Influencing factors
amplification screening
amplify
Screening method
Screening based on vector antibiotic resistance genes & corresponding selected drugs
Screening method for transforming Escherichia coli with plasmid vector carrying double antibiotic resistance markers
Carrier: Amp (resistant to ampicillin), Tet (resistant to tetracycline)
A foreign gene is inserted into one of the antibiotic resistance genes causing its inactivation
Two antibiotic plates to screen recombinants
step
①Positive selection
Contains antibiotics corresponding to non-inserted inactivated resistance genes
Transformant√
Transformant containing plasmid containing target gene √
Transformant containing recombinant target gene √
Non-transformant ×
②Negative selection
Antibiotics containing insertionally inactivated resistance genes
Non-recombinant transformants that do not contain the target gene√
Transformants containing recombinants of target genes×
Comparison of the two media → Selection: Transformants containing recombinants of the target gene
Screening using lacZ gene of lactose operon (blue-white spot)
Bacteria with a complete lactose operon can translate the LacZ gene encoding β-galactosidase → hydrolyze X-gal → blue
Foreign gene insertion → LacZ gene cannot be expressed → white
Required: No inactivated antibiotic resistance gene inserted → ampicillin culture medium
Transformant containing plasmid containing target gene: strain blue
Transformant containing recombinant target gene: strain white
Application of DNA chip to identify recombinants
Many specific DNA oligonucleotides/DNA fragments (probes) → fixed: preset position of the chip
Sample to be tested: label → hybridize with chip (principle of complementary base pairing) → detect hybridization signal & computer analysis
Screen based on target gene translation products (proteins, enzymes, polypeptides) [No marker gene or cannot synthesize probe]
protein gel electrophoresis
PAGE
Immunoassay
ELISA
Western blotting
Immunoprecipitation
solid-phase radioimmunoassay
Expression of target gene
Applications in the food industry: genetically modified foods
genetically modified microbial food
definition
Engineering bacteria
Features
application
(1) Improve the quality of food products
(2) Simplify the production process and shorten the production cycle
(3) Antibacterial and antiseptic preservation of food
(4) Improvement of food-grade enzyme production bacteria
(5) Production of functional ingredients for health food
(6) Rapid detection of food microorganisms
genetically modified animal food
Involved types
main application
Change livestock spawn rate;
Improve the nutritional composition of dairy products, especially for infants and young children
Production of human serum proteins, etc. through bioreactors.
genetically modified plant foods
definition
type
application
Improve the quality of food ingredients
Improve food production processes
Production of food additives & functional foods