9: DNA based technologies Flashcards
genome definition
the complements of genes that an organism contains. In humans very little of the DNA actually encodes genes for proteins (~30,000)
genomics definition
the study of genes, how they are arranged, how many, what are they similar to
proteome definition
the complement of proteins that an organism produces from its genome and post translational modifications. It is dynamic and huge!
proteomics definition
study of the proteome, how these proteins are expressed, when they are expressed, what is their function, how do they interact
DNA cloning definition
to separate a gene from a larger piece of DNA (such as a chromosome) and replicate it
Basic concepts of DNA to protein central dogma. how does each step start and end?
replication: DNA polymerase starts at origin of replication sequence and ends at replication termination sequence
transcription: starts at transcription start site bracketed by 5’ sequence. different polymerases use different promotes. ends at transcription termination sequence
translation: starts at an AUG sequence that is bracketed by ribosome binding site. ends at a stop codon
Basic process of DNA cloning
- select a cloning vector: must contain all the necessary elements to convince host to amplify, transcribe, translate
- PCR: select your DNA of interest
- Ligate: join the pieces of DNA
- Transformation: transfer the recombinant DNA construct into a host cell (E coli)
- select the cells with harbor the recombinant DNA
types of vectors and size of insert they can carry
Plasmids: naturally occurring, circular pieces of double stranded DNA. Design to carry genes that are advantageous to host, can be autonomously replicated, and kept small and simple for use as cargo ship. Insert must be small, <3kb
Bacteriophage: linear double stranded DNA molecules that can be packaged into the head of a bacteriophage. about 1/3 of its genome is non-essential, can be replaced by foreign DNA of around 15kb. use when large amounts of lengthy DNA are needed
Cosmids: hybrid plasmids with lambda phage cos sequence (behave kind of like bacteriophage). somewhat unstable and sometimes difficult to maintain. Large insert possible, around 40kb
BACs/YACs: bacterial/yeast artificial chromosomes. for very large inserts, 300kb and YACs up to 3000kb
cloning plasmids all have:
- replicator (ori)
- selectable marker, eg antibiotic resistance gene
- cloning site or multiple cloning site (MCS)
what is the relationship between transformation efficiency and plasmid size
inversely proportional, smaller plasmids transform more efficiently. keep both the vector and insert as small as possible
What do you need to make a PCR work?
template
primers
DNA polumerase
dNTPs
describe the PCR process
heat strands to 98°C to separate (denature). add the primers and cool to anneal. add DNA polymerase to catalyze 5’-3’ DNA synthesis (only gos in 5 to 3!). repeat the process about 25 times to amplify 10^6 fold
what are restriction enzymes and their use?
In order to insert DNA you need to cut the plasmid and insert at a defined site which matches and can be ligated together. This is done by restriction enzymes (endonucleases) which recognize and cut double stranded DNA at specific restriction sites (a 4-6 bp palindrome). Native purpose to cleave foreign invading DNA. Can generate sticky or blunt ends
how to find restriction sites
in vectors: provided by companies that sell vectors, called multiple cloning sites (MCS)
in DNA with gene of interest: plug sequence into online search engine to find.
what if there are no convenient restriction sites around the gene of interest?
- linkers/adapters
- PCR to add a restriction site: add an overhang to the primer that encodes a restriction site sequence, so when the sequence is replicated in now contains a restriction site
What does ligase do?
seals the DNA backbone using ATP in the process
what are the options for combining restriction enzymes to get the most reliable result?
Cutting with a single RE is not super reliable, it can insert backwards. Cut with 2 REs to avoid this. most to least reliable:
- clone with two sticky ends
- clone with one sticky end and one blunt end
- clone with two different but compatible ends (doesn’t match but still anneals, destroys palindrome)
- clone with two blunt ends, last resort option. not very good
how to make competent (willing to accept exogenous material) cells for transformation
two ways:
- chemically: add CaCl2 and heat shock
- electroporation: apply high voltage
why use selection markers?
selection markers ensure that only the bacteria that take up the DNA grow. put an antibiotic resistance gene in the plasmid and grow in media containing antibiotics so only the bacteria harboring the plasmid can grow
special considerations for designing protein expression proteins
expression vectors: contain necessary elements for transcription and translation of genes
must insert the correct direction (use MCS)
bacterial promoter and operator sequences (upstream)
genes encoding regulators for promoter and operator
ribosomal binding site (upstream)
termination sequence (downstream)
how to visualize your protein expression
use a fusion tag. encode green fluorescent protein in line with the gene of interest in the expression plasmid. when the protein is translated, the protein will include the fluorescence protein and can be visualized
how to use a fusion tag to aid in purification
include coding for a tag (like 6 His residues) in line with the gene of interest in plasmid. Grow cultures, induce synthesis, harvest cells, break open, centrifuge cell debris, and save supernatant which has soluble proteins. Use the biochemical properties of the tag as a method of selecting for the protein (affinity chromatography)
how to perform site directed mutagenesis on a plasmid
create PCR primers harboring the desired mutation. perform several rounds of PCR, producing DNA plasmids with the mutation incorporated. digest the parent DNA with DpnI restriction enzyme which cuts methylated DNA. Only the parent wild type DNA is methylated
what is a cDNA library and how is it made?
cDNA = copied DNA from mRNA template
library is a collection of random DNA pieces from an organism that is all cloned. Each single piece of the total DNA is harbored in a bacterial colony, Use southern blot to find which E coli cells have the desired DNA
9 pt 1 slide 24
describe southern blot
based on ability of a labelled DNA probe of a specific sequence to anneal to a complimentary DNA strand in the library. grow transformed colonies on agar, press nitrocellulose paper onto the plate. some cells from colonies stick to the paper. treat with alkali to disrupt cells and expose denatured DNA. add a radiolabeled DNA probe that will anneal to target DNA and expose which colonies have the target.
You can then transfer and amplify those colonies to collect target DNA
9 pt1 slide 25-27
how to design a probe for southern blot
probe should have complimentary sequence to target DNA. can use protein homology:
if you know that similar genes in other organisms contains a certain sequence, make a probe with that sequence for your gene.
if you know the protein sequence of the gene you can create a probe with matching DNA sequence. pick amino acids with minimal degeneracy and then account for possible variations at whatever variable sites remain. Order probes with all possible combinations
northern blot vs southern blot
northern: screening RNA
southern: screening DNA
both techniques are qualitative
what enzyme is responsible for making cDNA? how does it work
cDNA is made from a mRNA template via reverse transcriptase (RT). the mRNA template has a poly A tail (in eukaryotes) which we can anneal a synthetic oligonucleotide (oligo dT) primer to. RT will then create a complimentary yield with dNTPs. the template mRNA is degraded with alkali and DNA polymerase I makes new complimentary strand
summarize common enzymes used in molecular biology
type II restriction endonucleases: cut DNA at specific sites
DNA ligase: seals the backbone of 2 DNA strands
DNA polymerase: polymerizes strands of DNA using a template strand, 5 to 3
reverse transcriptase: copies an RNA template into a cDNA strand
polynucleotide kinase: adds a phosphate to the 5’-OH end of a polynucleotide to label it or permit ligation
alkaline phosphatase: removes terminal phosphate from the ends of DNA to prevent plasmids from self ligating and closing up
describe levels of protein function
phenotypic function: describes the effects of a protein on the entire organism
cellular function: the network of interactions engaged by a protein at the cellular level
molecular function: the exact biological activity of the protein
4 main paths for studying protein function
- sequence comparisons with genes and proteins of known functions (genomic, cDNA libraries, algorithms/databases)
- determining when, where, how much a gene or gene product is expressed (GFP fusions, 2D PAGE, DNA chips, RNA-Sep, QRT-PCR)
- investigate the interactions of the protein with other proteins (pull-downs, IPs, and yeast 2 hybrids)
- know down expression of a gene (RNAi and CRISPR)
describe using libraries and sequence homology to study protein function
use comparative genomics or BLAST search. Comparative can include ortholog or paralog.
ortholog: gene from DIFFERENT species, but have a clear sequence and functional relationship to each other
paralog: genes similarly related but from SAME species
describe 2D PAGE
proteins are loaded onto membrane strip with a pH gradient. proteins migrate on the strip according to their pI. apply the strip to top of gel and rung SDS PAGE so proteins migrate according to their molecular weight. Can then excise protein spots from gel and identify them using mass spec
describe mass spec
technique to identify a protein in a gel/solution. process of separation, digestion, detection, and identification to produce amino acid sequence.
Do NOT need to know what you are looking for. expensive special technique. quantitative??
describe Western
technique to identify a protein in a gel. separate protein samples on SDS PAGE and transfer onto PVDF or nitrocellulose membrane. block the membrane with neutral protein (BSA or milk). incubate with primary antibody specific to target protein. incubate with HRP-labeled secondary antibody specific to primary antibody. incubate the blot with chemiluminescent HRP substrate and expose to film
need to KNOW what you are looking for to make specific antibody. qualitative
describe the structure of antibodies
Y shape with two antigen binding site prongs (Fab region) and constant Fc region. IgG is most common, 70-75% of all human anitbodies.
how are specific antibodies produced
Primary antibody: prepare the antigen (target protein XYZ) and inject in rabbit to produce antibodies. purify the rabbit antibody (written as rabbit anti-XYZ). primary antibody uses Fab region to recognize
Secondary antibody: prepare the antigen (mouse IgG) and inject in rabbit to produce antibodies. purify the rabbit anti-mouse IgG (rabbit a-mouse). These are selected for those that recognize the Fc region (which is constant in mouse) so antibodies will recognize all mouse IgGs.
9 pt2 slide 8
describe DNA microarrays (DNA chips)
oligonucleotides representing segments of known genes are spotted onto a glass slide (10,000-65,000 spots) and labeled mRNAs or cDNAs from sample of interest are added. hybridization occurs between oligonucleotides on chip and complementary segments of supplied mRNA or cDNA. the labels show which genes are expressed in your sample
Cannot recover samples for cloning. IDENTIFICATION method only. only reports how much mRNA is expressed, NOT how much protein is expressed
9 pt2 slide 9-10
describe RNA-Seq
shows which genes are transcribed and how much. mRNA is copied to cDNA and shattered into fragments. sequence fragment ends and map reads.
IDENTIFICATION method only, cannot recover. only reports mRNA, NOT protein expression
describe QPCR
can quantify specific RNA transcripts. Reverse transcriptase transcripts RNA to cDNA, then PCR with special fluorescent probe (only fluoresces when bound to DNA due to forming a hairpin on itself). PCR can be used quantitatively by monitoring the progress of the amplification and determining when a DNA segment has been amplified to a specific threshold level.
must know what you’re looking for!
what is the idea behind pulldowns, IPs, and Yeast two-hybrids?
you can investigate your protein of interest by its interactions with other proteins. if it interacts with lots of a certain type of known protein, maybe it is also that type. guilt by association
describe pull down experiments
express a protein with a fusion tag to allow easy purification (like His6). This protein has a binding partner, you can purify the two bound proteins and then identify the unknown binding partner by mass spec or western etc. This will not work if the binding partner interacts with the termini containing the His tag. Tag can disrupt binding
what is IP
immunoprecipitation: an affinity resin that uses an antibody
describe the elements used in a yeast two hybrid system
reporter gene measures transcription amount (eg fluorescent protein). promoted by Gal4p
bait protein bound to Gal4p DNA binding domain
fish/prey protein on transactivation domain on Gal4p.
Gal4p is a protein that activates certain genes in yeast by binding DNA element and increasing RNA polymerase and transcription. important to know the DB (DNA binding) and AD (transactivation) domains. they work separately as long as something can bring them together
yeast two hybrid system set up and read out
- synthesize constructs from a proteome library. create the bait attached to Gal4p BD. create prey with Gal4p AD attached.
- screen proteome library for potential interactions. when bait and prey interact they bring the BD and the AD domains close and therefore promote transcription of the reporter gene and increase signal.
9 pt2 slide 18
idea behind RNAi and CRISPR
investigate protein function by removing it in an organism and seeing if its absence has the expected effect
describe RNAi
knocking down gene expression. purchase siRNA which RISC complex in cells will bind one strand of. this pairs with the target and makes a complex which exonuclease degrades. does not completely remove the expression, just knocks it down
describe CRISPR
regulates gene expression using Cas9 to bind the guide RNA and have nuclease activity. the guide RNA has target sequence coding.
organisms used for recombinant protein expression
most commonly bacteria like E coli. vectors are optimized for particular organisms, like the pET series of vectors will express in bacteria only. mammal, insect, yeast, algal, and cell free also possible
what role does recombinant protein expression have in vaccine development
9 pt2 slide 23
can insert DNA vaccines, RNA vaccines, vector vaccines, into a cell for expression of the modified viral proteins. Then you are able to build up immune response to the viral proteins
