Chapter 19- Molecular Genetics and Biotechnology Flashcards
1
Q
Innovations of Molecular Genetics
A
Recombinant DNA Technology
Polymerase chain reaction (PCR)
DNA sequencing
Genome editing systems
2
Q
Limitations?
A
- Genes are tiny
- there are thousands of them in every single cell
- nucleotides are not visible
- there is no obvious beginning or end to each individual gene
3
Q
Recombinant DNA Technology
A
- Major revolution in molecular genetics (1973)
- techniques for locating, altering, combining, and studying DNA fragments/genes
- powerful tool for genetic engineering of organisms
- combining DNA from 2 different sources
4
Q
Restriction Enzymes
A
- recognize specific sequences in DNA and make double stranded cuts at restriction site
- 4-8 base pair cutters (they eat up the named number of base pairs)
- cohesive ends (sticky ends) allow for connection via “gluing” complimentary ends
- blunt ends
- less specific and smalles bp cutters will cut more stuff
Recombinant DNA Technology
5
Q
Visualizing DNA
Recombinant DNA Technology
A
- gel electrophoresis
- negative to positive, pulls negatively charged DNA molecules done through towards the positive charge
- ethidium bromide is used as a stain to make the DNA fluoresce
- northern blotting for DNA
- southern blotting for RNA
- western blotting for proteins
6
Q
Cloning Vectors
Recombinant DNA Technology
A
- stable DNA molecule to which a foreign DNA fragment can be inserted or attached for introduction to a new cell
- typically bacterial plasmids, you take the plasmid and put it in a bacteria through recombination allowing for free replication in another organism
- cloning vectors allow for replication, have a selectable marker, and have one or more unique restrictive sites that the DNA fragmeny can be inserted into
7
Q
Blue-White Screen
Cloning Vectors
A
- loking for live bacteria in treated media
- lacZ lets galatizidase in wher foreign DNA would be inserted, looking for interrupted lacZ (white colonies)
- X gal, bacteria with intact lac Z will cleave this making the colonies appear blue
8
Q
Applications of Recombinant DNA Technology
A
- disease therapeutic genes in humans
- herbicide/pesticide/drought resistance
9
Q
Polymerase Chain Reaction
Amplification of DNA
A
- denaturing
- annealing
- elongation
10
Q
Dieoxy sequencing (Sanger)
Sequencing DNA
A
- use sample from PCR and dye terminator (ddNTPs) that lack 3’ OH group
- do another PCR w/ ddNTPs rather than dNTPs
- when ddNTP is incorporated it stops sequencing of the new strand
- generates many fragment sizes
11
Q
Sequencing by Synthesis
Sequencing DNA
A
- break DNA into small fragments
- washed aeros flow cell in sequences where they get strands
- primers and polymerases added
- synthesize and flourescent nucleotides added
- computer reads every single nucleotide, and after reading the flourscent is pulled off and replaced with the normal nucleotide
- many read at once
12
Q
Third Generation
Sequencing DNA
A
- Pac Bio
* aumate
* long read sequencing
* ~10,00-200,000 bps
* time consuming
* more expensive - Oxford Nanopare
* long read sequencing
* higher error rate
* size of a thumb drive
* can be used in the field
* less expensive
13
Q
DNA Fingerprinting
Sequencing DNA
A
- identify individual by unique DNA nucleus
- microratteletes/ short tendon repeats (SRTs)
- primers sit on either side of STRs sequence from both sides
-homozygotes have large peaks
-heterozygotes have smaller peaks
14
Q
CRISPR-Cas9
Gene Editing
A
- Naturally occurring in bacteria and used for immunity and protection against viruses and other invading DNA elements
- can be engineered to find unique sequences in the genome for editing (more specific than restriction enzymes)
- CRISPR RNA and Tracer RNA combine with cas9 to make effector complex
Ethical issues here
15
Q
Site-directed Mutagenesis
Controlling Gene Function
A
- forward genetics is phenotype –> genotype
- reverse genetics is genotype –>phenotype
16
Q
Transgenics and Knock-outs
Controlling Gene Function
A
- can add in genes that are not normally there
17
Q
RNAi
Controlling Gene Function
A
- small interfering RNAs
- can control peptide sequencing
- way of controlling gene function
- a way of silencing genes without editing the actual gene
siRNAs can combine with proteins to form RNA-induced silencing complex (RISC) that cleaves mRNAs and prevents their translation
18
Q
Biotechnology and Beyond
A
- is a rapidly growing field
- biotech is an all-encompassing field with practical applications
- roots in molecular genetic techniques and utilizes them in different ways for different practical purposes
- ethical and societal implications (needs to be monitored appropriately)
