Genetic technology Flashcards
1
Q
What are three ways of transferring genes?
A
- using restriction endonuclease to extract gene
- making gene from mRNA from reverse transcriptase
- artificial synthesis of gene from nucleotides using computer programming
2
Q
How does restriction endonuclease work?
A
- derived from prokaryotes/bacteria
- cuts DNA at specific sites
- can be palindromic (blunt) or staggered (sticky) ends
- destroys viral DNA in bacteria
- same restriction enzyme used for vector
3
Q
How is DNA made from reverse transcriptase?
A
- add DNA primer to mRNA
- mRNA is a template
- DNA strand synthesized by reverse transcriptase
- cDNA formed
- DNA polymerase uses cDNA to create double strand
4
Q
Why are plasmids good vectors?
A
- small: easily taken up by the cell
- can be cut by restriction endonuclease: has specific sites
- replicate independently from each other: so a large amount of DNA is transcribed
- contain marker genes: such as antibiotic resistance genes, so can be recognized
- circular: makes them stable
5
Q
How are plasmids inserted into bacteria?
A
- transformation
- plasmid and bacteria bathed in ice-cold calcium chloride solution
- incubated at 40 degrees C
- bacteria given electric shocks
- this makes membrane porous
- so plasmids can enter
6
Q
What are the five main enzymes used in gene technology?
A
- DNA ligase
- DNA polymerase
- Restriction enzyme
- Reverse transcriptase
- Cas9
7
Q
What are the three main vectors used in gene technology?
A
- Plasmids
- Viruses
- Liposomes
8
Q
What is a promoter?
A
- length of DNA containing binding site for RNA polymerase
- controls gene expression and transcription
9
Q
Why are promoters needed for gene transfer?
A
- promoters initiate gene expression/transcription
- binding site for RNA polymerase
- otherwise gene has to be inserted near existing promoter
- can disrupt gene expression
- precise position of promoter in eukaryotes
10
Q
What are marker genes?
A
- gene transferred with desired gene
- to identify cells that have been altered and contain rDNA
11
Q
What are some advantages to marker genes over antibiotic resistance genes?
A
- easy to identify/screen
- more economical
- some antibiotics are no longer effective (bacteria will still grow)
- resistance gene can be passed to other bacteria
12
Q
Outline the process of gene editing
A
- form of genetic engineering
- involves deletion, insertion or replacement of gene
- Cas9: cuts DNA at specific points determined by the base sequence of a molecule of guide RNA
13
Q
What is PCR?
A
- amplification of DNA
- only small sample of DNA needed
- rapid
- Denaturation: DNA separated into two strands at 95 degrees
- primer added
- Annealing: 60-65 degrees
- complementary base pairing between primer and strand
- Extension: DNA strand synthesized at 72 degrees
- done by DNA/Taq polymerase (which is stable and can be reused)
14
Q
Why is Taq polymerase used in PCR?
A
- high optimum temperature/heat stable
- does not need replacing each cycle
- process is more efficient/faster than using DNA polymerase
15
Q
Outline the process of gel electrophoresis:
A
- DNA collected from root of hair or saliva
- DNA amplified by PCR
- DNA fragmented by restriction endonuclease
- enzyme will cut close to VNTR regions
- DNA loaded into agarose gel by micropipette
- DNA must be close to negative electrode
- gel submerged in electrolyte/buffer solution
- electrical current applied to tank
- DNA phosphate groups give negative charge
- attracted to anode
16
Q
What affects the speed of DNA strands in gel electrophoresis?
A
- Shorter strands move faster
- More negative strands move faster
17
Q
How is genetic fingerprinting used in detection of DNA?
A
- after gel electrophoresis, DNA fragments are transferred to absorbent paper
- DNA probes complementary to VNTR sequences are added
- Probes have fluorescence dye
- UV light used for fluorescent detection
- banding pattern created
18
Q
What are VNTR regions?
A
- variable number tandem repeats
- only vary by number of repeats (exception: identical twins)
- VNTR’s with more repeats are longer
19
Q
What are microarrays used for?
A
- used to identify active/switched on genes
- transcription of a gene produces mRNA
20
Q
Outline the process of microarrays:
A
- ssDNA act as probes
- probe is complementary to the DNA being tested for
- many copies of one type of probe placed in each cell
- single stranded DNA made from mRNA
- DNA labelled with fluorescent dyes
- DNA hybridizes with probes
- unbound DNA washed off
- laser/UV light used to detect presence of DNA/hybridization
21
Q
What is bioinformatics?
A
- large collection of biological information/DATABASES on a computer software
- information can be about organism genomes
22
Q
How are bioinformatics used?
A
- primary structure of proteins can be compared
- 3-D/tertiary structures can be visualized
- genes can be recognized
- protein function can be identified
23
Q
What are the advantages of bioinformatics?
A
- cheaper
- less equipment needed
- faster
- fewer ethical concerns regarding humans and animals
- can have different changes to model
24
Q
What are the disadvantages of bioinformatics?
A
- difficult to assume that protein actually functions
- safety: side effects cannot be observed (context of drug)
- dosage cannot be determined (context of drug)
25
How is a gene containing human insulin inserted into a plasmid?
- **mRNA taken from B-cells** of pancreas
- cDNA made by **reverse transcriptase**
- **DNA polymerase used to make cDNA double stranded**
- **sticky ends created**
- **plasmids** are cut with **restriction enzyme**
- **complementary sticky ends** added
- cDNA mixed with plasmid
- **DNA ligase** seals nicks in sugar phosphate backbone
26
What three human proteins can be produced from genetic engineering with rDNA?
- Insulin (plasmids + bacteria or from pigs)
- Factor VIII (hamster cells)
- Adenosine Deaminase (viral vector + bacteria or from cattle)
27
What is genetic screening?
- **analysis of a person’s DNA** to check for the presence of one or more alleles associated with the disease
- testing **embryo/adult/fetus**
28
What diseases can be screened?
- Huntington’s
- Cystic fibrosis (CF)
- Breast cancer genes
29
What are the advantages of genetic screening?
- **informed reproductive decisions** can be made
- abortion choice
- embryo can be discarded
- **preventative measures/early treatment** options can be taken (breast cancer)
30
What are the techniques for prenatal diagnosis?
- **Amniocentesis**: amniotic fluid taken
- **Ultrasound** scanning
- **Chronic villus sampling**: small part of placenta removed with needle
- screening
31
Outline the aims of gene therapy:
- to **insert a normal/functional _allele_**
- to **obtain a normal/functional protein**
- to **reduce symptoms** of disorder
- **enhance cellular functions**
- **increase quality of life**
32
What diseases use gene therapy?
- SCID (faulty ADA gene)
- Leber congenital amaurosis (inherited blindness)
- CF
33
What are the social and ethical implications of gene therapy?
*pros*
- treats **cause** not symptoms
- **less time consuming** than other treatments
*cons*
- **side effects**
- **expensive** treatment
- follow up treatments
- **designer babies**
34
What are the advantages to using insulin produced by gene tech?
- **identical** to human insulin
- less chance of **side effects**/rejection
- **fewer ethical** concerns
- reliable supply available
- cheap to produce in large volumes
- **rapid** response
- useful for those with **animal insulin tolerance**
- less risk of transmitting disease
