recombinant DNA technology

Question 1

What is a gene?

Answer 1

• the functional units of chromosomes, determine specific characteristics by coding for specific proteins. Traits are the variations of those characteristics. For example, hair color is a characteristic with traits that are blonde, brown, or black, and many colors in between
• the basic physical and functional units of heredity

Question 2

Define and distinguish the terms ‘genetic engineering’ and ‘recombinant DNA technology’?**

Answer 2

• genetic engineering- the alteration of an organism’s genotype using recombinant DNA technology to modify an organism’s DNA to achieve desirable traits. The addition of foreign DNA in the form of recombinant DNA vectors generated by molecular cloning is the most common method of genetic engineering.
• recombinant DNA technology- using enzymes and various laboratory techniques to manipulate and isolate DNA segments of interest. This method can be used to combine (or splice) DNA from different species or to create genes with new functions. The resulting copies are often referred to as recombinant DNA.
• All in all, genetic engineering is a broader term used to describe all the techniques that can be used for the alteration and modification of DNA, whereas Recombinant DNA Technology is one of the techniques of genetic engineering that is used to produce recombinant DNA.

Question 3

How are genes sequenced?**

Answer 3

• gel electropherisis
• create a DNA library (clone fragmens into a plasmid vector)
• sequence each clone
• align overlapping sequences and assemble the genome
• can compare individuals to eachother

Question 4

What is biotechnology?

Answer 4

• use of biological agents for technological
  advancement
• utilizes biological systems, living organisms or parts of this to develop or create different products.
• ex. the development of insulin, crispr

Question 5

Explain molecular and reproductive cloning

Answer 5

• molecular cloning: technology fundamental to molecular biology and biotechnology that uses living cells (ex. bacteria, yeast) to make many copies of a DNA sequence of ineterst. plasmids are essential. produces recombinant proteins. create multiple copies of a specific DNA fragment, such as a gene or a regulatory sequence, and to transfer it into a host organism. can be used in gene therapy, gene anaylsis, biomedical applications
• reproductive cloning: the deliberate production of genetically identical individuals.ex. dolly the sheep. creating an organize that is genetically identical to donor organism

Question 6

Describe biotechnology uses in medicine and agriculture

Answer 6

• crispr.ex. allergies, alter genome
• gene therapy: can be used to provide genes to individuals who dont have a functional one. ex. build plasmids containing a normal version of the gene that’s nonfunctional in cystic fibrosis
• biomedical applications: vaccines. insulin
• gene anaylsis: build dna to help understand how normal genes work
• gmo crops to be resistant to pesticides and grow bigger

Question 7

Describe the key concepts and principles of molecular cloning

Answer 7

• fundamental to molecular biology and biotechnolgy
• uses living cells to make many copies of a DNA sequence of interst
  workflow: 1. select plasmid –> 2. target DNA isolation –> 3. create recombinant DNA –> 4. propogate recombinant DNA in bacteria (or another sutiable host) –> 5. screen and select bateria that express your recombinant DNA –> 6. isolate recombinant DNA for further verification and experimentation

Question 8

cDNA synthesis

Answer 8

• copy dna
• synthetic DNA that has been transcribed from a specific mRNA through a reaction using the enzyme reverse transcriptase. mrna is isolated and turned back into DNA
• useful for cloning and expressing specific genes
• create cDNA libraries, enabling them to study sequences specific to a given tissue and develop shareable databases
• contains only the expressed genes of an organism, excluding introns (non-coding regions) and regulatory sequences. This makes cDNA particularly useful in genetic engineering, molecular biology, and gene expression studies.

Question 9

What is PCR?

Answer 9

• polymerase chain reaction
• DNA amplification in a test tube
• a technique used to amplify small segments of DNA (specific sequences)
• makes billions of copies of a specific DNA fragment or gene, which allows detection and identification of gene sequences using visual techniques based on size and charge.

Question 10

what is restriction digest

Answer 10

• technique used in molecular biology to cut DNA into smaller fragments using specific enzymes known as restriction enzymes
• Restriction enzymes, also called restriction endonucleases, recognize specific, short nucleotide sequences in the DNA and cleave the DNA at or near these sites.recognize unquie sequences
• combining a DNA sample with one or more restriction enzymes in a reaction buffer that provides the necessary conditions for the enzyme activity. then incubate. afetr digestion, dna fragments can be separted using gel electropherisis and analyzed

Question 11

what is bacterial transformation.

Answer 11

• bacteria take up foreign DNA from their surrounding environment. This genetic material is often in the form of plasmids,
  -Once inside the bacterial cell, the foreign DNA can be incorporated into the host genome or can remain as an independent plasmid
• f the foreign DNA confers a new trait, such as antibiotic resistance or the ability to metabolize a new substrate, the recipient bacterium can express this trait and pass it on to its descendants.

Question 12

Define genome editing

Answer 12

• a menthod used to alter an organism’s DNA by leveraging the cells DNA repair mechanisms
• during cell division, if there is an error, these repair mechanisms will stop division and fix. they will correct mutattions,insert dNA sequences (ex. GFP tagging), and remove DNA sequences (ex.create knock outs = loss of protein function)
• uses recombinant enzymes (endonucleases) to cleave and alter DNA sequences. tools include: zinc finger nucleases, transcription activator-like effoector nucleases (TALENs), and CRISPR

Question 13

Compare and contrast the mechanisms by which different genome editing tools edit DNA

Answer 13

ZNFs and TALENs use FOK1. CRISPR uses bacterial immunity

Question 14

Explain how CRISPR is used naturally by cells and in the context of biotechnology and healthcare.

Answer 14

• Bacteria capture and store short, unique fragments of viral DNA in their CRISPR arrays during past infections.
• If the same virus attacks again, the bacteria use these stored sequences to generate RNA molecules (CRISPR RNAs or crRNAs) that guide Cas proteins to the specific location on the viral DNA.
• Cas proteins then act like molecular scissors, cutting the viral DNA and preventing the infection.

Question 15

what is knockout

Answer 15

a gene that is no longer functional. knocks out the function

Question 16

endonuclease

Answer 16

nucleotides that cut DNA. ex. restricition enzyme

Question 17

what are different DNA repair processes

Answer 17

• depends on the damage to DNA

single stranded break:
- base excision repair: if base is damaged, it is repaired; DNA glycosylase removes the damaged base; short-patch or long-patch repair then fills the gap
- nucleotide excision repair: cut nucleotide out and uses the intact strand as a template to fill in the gaps

double stranded breaks (no template to reference), :
- homologous recombination: uses homolog to fix. homologous partner from cell division where two copies are avalible. only avalible in some points of cell division. S-phase and after
- non homologous end joining: no template avalible. 2 broken ends ligate back together. could lead to loss of DNA. this could have a big impact if it occurs on a protein

Question 18

FOK1

Answer 18

• a non-specific restriction enzyme that can ony function as a dimer
• will cleave non discriminantly
• doesnt only cut at specific points
• dimerization of FOK1 results in one douvle stranded cut
• to replace with new DNA sequence, you must use four ZFNs/TALENs

Question 19

how do ZFNs and TALENs use FOK1

Answer 19

ZF:
- ZF domain binds 3 nucleotides at a time
- have 3-4 ZFs ina ZFN
- use the ZFN that has th sequence that you want
- ZFn directs FOK1
- NEEDS 2 FOK1, only 1 will not work

TALEs:
- function similarily to ZFs, but TALEs bind to 1 nucleotide at a time
- more easily customizale; endless number of sequences
- cuts in 1 line

• Zfs and TALEs directs but dont cut. tells FOK1 where to go and binds to DNA

Question 20

CRISPR** how detailed???

Answer 20

• CRISPR “spacer” sequences are transcribed into short RNA sequences (“CRISPR RNAs” or “crRNAs”) capable of guiding the system to matching sequences of DNA. When the target DNA is found, Cas9 – one of the enzymes produced by the CRISPR system – binds to the DNA and cuts it, shutting the targeted gene off.
• provide a template and we can sunsitute our template in to fill nucleotides

Question 21

CRISPR for sickle cells

Answer 21

-sickle cell is a mutation in the beta globin gene. is not present at infancy. present after around 6 months
- crispr can be used by extracting stem cells and turning off the BCL11A gene.the stem cells are gentically modified and returned to the person. this make normal red blood cells

Question 22

ethics of CRISPR

Answer 22

• cell based therapy can leverage the power of gene editing to correct potentially disease-causing mutation in the genome (ex. cancer, HIv, literally anything caused by DNA mutation)
• should embryonic/ germline gene editing be allowed??
• genetically modify animals??
• healthcare: precision and personalized medicine
• impacts on/adaptions to climate change

Question 23

1. select your (genetically engineered plasmid)

Answer 23

• not all plasmids are created equal
• plasmids are used because they have features essential for cloning such as (we will make lasmids have the elements that we want): multiple cloning sites (we induce this; site in plasmid that is recognized by multiple restriction enzymes, promoter sequence (DNA sequence responsible for regular rate of transcription), origin of replication, antibiotic resistance gene (bacterial selection) (plasmids have this!! plasmids are how resistance is transmitted to other genes), selectable marker (mammalian selection
• plasmids are smaller sequenes of DNA. non-essential gene for normal living conditons are found on palsmids
• which restriction site(S) will you use? choose a sequence NOT in target sequence. or else youll cut it

Question 24

2. Target DNA isolation

Answer 24

sources of DNA
- have to get the DNA from somehwere
- ex. human kasio DNA sequence can be aquied from: tissue biopsy, cultured cells derived from human tissue, or from another expression vector
- the source you use depends on the context in which you are working

Genomic or cDNA(complementary dna)
- reverse transcription: from DNA to mRNA to protein
- we want to isolate mRNA and turn it back into DNA
- mRNA only has poly A tail; no interons (pre mRNA has introns). the inrons are cut out by alternative splicing. cDNA has no introns so by using this we can skip alt splicing since its tie consuming

Create and amplify DNA
- reverse transritas reads MRNA and makes cDNA
- PCR amplifies cDNA
- final number of copies of cDNA: severla million to billions of cDNA molecules

Question 25

3. create your recombinant DNA

Answer 25

-PCR sequence of interst. resriction sites are added onto the primers. the restriction enzymes cut out ends. need to introduce sites or else it wont cut anything
- digest PCR amplicon and clong vector
- ligate and inesert into the vector –> expression construct. ligase glues the fragments together
- increased ration of inserts to vector to prevent it from resealing itself (more inserts)
- the insert is forced into the gap

Question 26

4. propagate recombinant DNA in bacteria (or another suitable host)

Answer 26

• force plasmids into bacteria through heat shocking
• put the DNA from ligation into testtube and it goes into bacteria
  -the bacteria with the plasmid is called transformed
• use bacteria bc they have short replication time

Question 27

5. screen and select bacteria that expresses your recombinant DNA

Answer 27

• select the bacteria with the plasmid
• introduce antibiotic to bacteria colony with the plasid
• recall that the plasmid has antibiotic resistance

Question 28

6. isolate recombinant DNA for further cerification and experimentation

Answer 28

• take broth and anibiotic and the plasmid is produced
• in case of kaiso, implant into pseudopregant mice

Question 29

how else is recombinant DNA used?

Answer 29

• create antibodies
• recombinant momcolonal antibodies (ex. herceptin, treatment for He2 breast cancer)
• recombinant vaccines
• human genome sequencing

Question 30

Explain the application and significance of molecular cloning in the context of genetic engineering and biotechnology.