Lecture 14/15: DNA Technologies and Genomics Flashcards
Biotechnology
Any technique applied to biological systems to manipulate processes for a specific purpose (everything in a cell)
Branches into
- DNA technologies
- Genetic Engineering
Purpose: We want to either: fix something OR figure something out
DNA technologies
- isolate, purify, analyze, and manipulate DNA sequences
(only DNA)
Genetic Engineering
- uses DNA technologies to alter genes for practical purposes
How do we know we dont get enough DNA for biotechnology?
- because we know 2 alleles aren’t enough we have to PRODUCE DNA
CLONE DNA
Cloning DNA
(PART 1)
1) isolate genomic DNA with gene of interest from cells and cut the DNA into fragments
2) Cut a circular bacterial plasmid to make it linear USING RESTRICTION ENZYMES to cut at specific nucleotide sequences
3) Insert the genomic DNA fragments into plasmid to make recombinant DNA molecules. Recombinant DNA is DNA from 2 different sources joined together
= recombinant DNA molecules are recombinant plasmids
DNA LIGASE WILL CONNECT THEM
4) Bring plasmids into bacterial cells and let them grow and divide w/ same plasmid (REPLICATION + DNA AMPLIFICATION)
5) Identify bacterium containing the plasmid w/ gene of interest inserted into it, grow bacteria in culture to produce large amounts on plasmid
Restriction Enzymes
= Endonucleases (cut nucleic acids within chromosomes)
- Eukaryotes don’t have them, bacterial cells however make them to fight against viruses
*Cut DNA at specific sequences in restriction sites
* The enzyme will cleave to make symmetrical restriction site w/ sticky ends on unpaired bases = restriction fragments
*stick them together and seal nicks with ligase = RECOMBINANT DNA
Plasmid Cloning Vectors
what are they engineered to contain
- bacterial plasmids that are engineered to contain
- Gene of Interest
- Sorting Genes
Ex.
1) lac-z gene (lac operon)
2) antibiotic resistance gene
= you MUST have these genes in vector because the cut will be in the lac-z gene so we can differentiate between white and blue colonies
CLONING DNA
(PART 2)
Q: How can we obtain our gene of interest?
A: You have 3 plasmids
1) w/ gene of interest
2) w/o gene of interest
3) w/ no inserted DNA fragment
(some will pick up recombinant or non recombinant and some may not grab ANY plasmid)
via transformation
= recombinant plasmids (from 1 and 2)
- antibiotic resistance genes can grow on agar plate
= non-recombinant plasmids (from 3)
= 1 no plasmid, can’t grow on agar plate
How do we know which colony has our gene of interest
a transformed bacteria grow on medium containing antibiotic (that bacteria is resistance to) because of amp gene on plasmid
- blue colony contains bacteria w/ a nonrecombinant plasmid; the gene is intact
- white colony contains bacteria with recombinant plasmid (vector + DNA fragment)
Untransformed bacterium can’t grow on medium w/ antibiotic, and so its not transformed w/ a plasmid
How do we know which white colony has our gene of interest
X-gal=substrate for B-galactosidase
= when hydrolyzed turned into a blue colour
DNA hybridization
DNA probe
- uses nucleic acid probe to identify gene of interest in set of clones
= complementation
Protocol:
- plasmid based library in plates
- growth medium containing antibiotic (all transformed + recombinant)
- nylon membrane: 1) lyse cells 2) denature DNA (becomes single stranded DNA)
- it’ll light up when u bind to target gene
= identify and isolate colony
DNA libraries
1) GENOMIC LIBRARY
- collection of clones containing every sequence in a genome
(bacterial cells/clones)
= such clones has a gene within genome
- library of different bacterial cells w/ diff genes of interest
- from genome of eukaryotic cells
= GENOMIC LIBRARY
2) COMPLEMENTARY DNA LIBRARY
- DNA sequences made from expressed RNA
= only genes that are actually being expressed which are actually transcribed because we look for mRNA
Making cDNA
- only includes the genes that are actually expressed
- this is how bacteria make clones
PROCESS
- we want to convert single mRNA into double DNA for cloning (RNA can’t be directly cloned
- Use reverse transcriptase (from retroviruses) to make a single stranded DNA that is complementary to the mRNA
- Degrade the mRNA with an enzyme and use DNA polymerase to make a second DNA strand complementary to the first
= cDNA
How can we make lots of DNA copies without using cells
PCR
x30 cycles=making copies of DNA of interest
1) denaturation
- separate the 2 strands w/ heat to break H bonds
2) annealing
- attach primers to templates
3) replication
- we need DNA polymerase from an organism that can withstand heat
(tag dna polymerase)
- group of prokaryotic organisms found living in hot springs…use and replicate their DNA because that’s the only way we can continue PCR
Agarose Gel Electrophoresis
Prepare the agarose gel with a DNA stain.
Load DNA samples into the wells.
Apply an electric current to separate DNA fragments by size.
Visualize DNA bands under UV light.
- technique where DNA, RNA, or proteins molecules are separated in a gel subjected to an electrical field
Restriction Mapping Linear DNA
- allows you to determine restriction sites for restriction enzmyes
DNA technology to detect genetic diseases
example: SICKLE CELL
Normal allele: 3 restriction sites w/ 2 fragments
Mutated Allele: 2 restriction sites w/ 1 fragment
= RESTRICTION FRAGMENTS LENGTH POLYMORPHISMS (RFLP): different fragments from some part of genome
How can RFLPs be detected
a) southern blot analysis: uses electrophoresis, blot transfer, and labelled DNA probes to identify RFLPs that match the B-globin gene
b) PCR and then electrophoresis
DNA technology for DNA fingerprinting
- Each person has their own unique set of DNA sequences= DNA fingerprint (nothing to do w/ ur actual fingerprint)
- Each locus has short tandem repeats (STR) that are found on our chromosomes and are repeated nucleotide sequences
# of STRs vary person-to-person - used in forensics and ancestry
= Variable number of repeats
Use of STRs in criminal cases and in paternity testing
- looking at STR markers to find the number closest to the DNA found on a victim or comparing dads DNA to child and mom
Why are we comparing it w/ mom too?
- bc remember baby will have 1 from mom, and 1 from dad
why can we never say 100% yes to paternity testing
- because another individual may have extremely similar or identical STRs such as an identical brother or twin
Transgenic Organisms
Transgenic organisms
- modified to contain genes (transgene) from an external source
Genetic Engineering of Bacteria
- put the trans gene in an expression vector that has a promoter for production of transgenic proteins
- We are using cDNA and not genomic library because bacteria don’t have introns *
Genetic Engineering of Animals
mouse embryo example:
- germ lines are taken from a mouse embryo
input transgene - pure population of transgenic cells
INPUTTED INTO:
- inject transgenic cells into embryo
- implant embryo into surrogate mother to produce babies w/ or w/o transgenic cells, when they produce offspring it will create genetically engineered offspring where all cells are transgenic BECAUSE SPERM/EGG CONTAIN TRANSGENE
Stem cells of animals
- Stem cell: unspecialized cell that can reproduce indefinitely and differentiate into various specialized cells
a) EMBRYONIC STEM CELLS
- isolated from early embryos at the blastocyst stage
- can differentiate into any cell types (pluripotent)
b) ADULT STEM CELLS
- replace non reproducing specialized cells
= can only be a certain group of cells
Human Gene Therapy
Gene therapy= alteration of individuals genes
- may treat disorders with one defective gene
Provirus: eukaryotic virus injecting itself into a eukaryotic genome
- viral DNA carrying the normal allele inserts into chromosome
= Patient produces normal protein
Somatic Gene Therapy (Humans)
Very difficult processes
- therefore, at times trying to fix 1 issues has lead to the development of another
- all cells except for germline and gametes because you don’t want it to carry on
Animal Genetic Engineering
a) Pharm animals produce proteins for humans through milk or other biological processes
- in milk: harmless extraction
= protein will only be made in mammary glands during lactation
b) Cloned mammals produced by implantation of diploid cell fused with denucleated egg cell
= low cloning success rate
= increased health defects in clones
= gene expression regulation abnormal
i.e. cloning a plant vs dog, dog is more complex so will be harder therefore, lower success rates
Dolly
- first genetically cloned animal
- we used 3 sheep total
HOW DID WE GET DOLLY?
1) adult white face and adult black faced (both females)
- diploid cell was isolated from mammary gland of white faced and put into the nucleus from unfertilized egg of black faced
= mammary gland cell was fused with enucleated egg cell (2n into egg)
Then…
- these cells were implanted into the black-face uterus and the embryo developed in surrogate (dolly), all of Dolly’s DNA was mitochondrial
Plant Genetic Engineering
- Isolating the desired gene.
- Inserting it into a vector (e.g., plasmid).
- Transforming plant cells via methods like Agrobacterium infection
- Selecting transformed cells and regenerating them into plants.
GMOs
- question about how ethical it is
yellow pig: transgene fluorescent gene
ear in mouse: not transgene
GMO rice: w/ carotene used in places w/o balanced diet
makes plants pest resistant
GENOMICS
characterization of whole genomes (structure, function, evolution)
a) sequencing: determine the nucleotide sequence of a genome
b) annotation: analyzes specific regions of nucleotide regions
c) functional genomics: studies functions of genes and other parts of the genome
d) comparative genomics
studies how genomes have evolved by comparing genomes of various organisms
Dideoxy (Sanger) method
- random insertion of dideoxyribonucleotide stops synthesos
- separate different sized DNA fragments with an electropheotric gel
- different lengths of DNA replicates, can be run through gel
- based on fluorescence, read DNA sequence 5’ to 3’
Genomic Sequencing
Whole-Genome shotgun method
1. DNA purification
2. DNA fragmentation
3. Amplification of fragments
4. Sequencing each fragment
5. Assembly of fragments into genome sequences
Human Genome
Human genome project
- revolutionizing biology and evolutionary understanding
Transcriptomics
a) Transcriptome: complete set of transcripts
b) Transcriptomics: study of transcriptome
Proteomics
Proteome
- complete set of proteins expressed by genome
- larger than genome in eukaryotes
Proteomics: study of proteome to determine
a) structure
b) location
c) interactions w. proteins + macromolecules
d) functions
Systems Biology
composition: genomics, transcripomics, protoenomics=system
a) studies organisms as a whole
- investigates networks of genes, proteins, and biochemistry
b) combines genomics and proteomics w/ response to environment
c) interactome=sum of all interactions of all gene products in a cell
genetic engineering in plants w/ Ti plasmid, and their purpose
Genetic engineering in plants uses the Ti plasmid (from Agrobacterium tumefaciens) to transfer desired genes into plant genomes, enabling traits like disease resistance, herbicide tolerance, or improved nutrition.
dideoxy method for DNA sequencing
modified nucleotides (dideoxynucleotides) that lack a 3’ hydroxyl group, causing DNA synthesis to terminate at specific positions, allowing the determination of the DNA sequence by analyzing fragment lengths.
Whole-genome shotgun method
The whole-genome shotgun method is a sequencing approach that involves randomly breaking an organism’s DNA into fragments, sequencing them, and assembling the overlapping sequences to reconstruct the entire genome.
DNA vs protein microarrays
DNA :measure gene expression by detecting mRNA levels,
PROTEIN: analyze protein interactions, abundance, or activity.
