bio 207 6 and 7
what are the challenges of molecular genetics
- challenge of scale (very thin and no physical features that mark genes)
-challenge of numbers (1 gene- 1/millionth genome, some genes ecpressed in small fraction of cells)
what are people able to do now in molecular genetics (5)
locate a gene/ DNA sequence, remove/copy DNA sequence, Visualize DNA, store new DNA sequences, Edit any genome
what does recombinant DNA allow you to do
locate a gene/ DNA sequence, remove/Copy DNA sequence, Visualize DNA, store new DNA sequences.
what is recombinant DNA (rDNA)
technique in molecular genetics where scientists combine DNA from two sources to a create new DNA. this is usually done using a vector like a plasmid.
what is recombinant also reffered to as
genetic engineering
what is a plasmid
small circula double stranded DNA that can naturally exist in bacterial cells along side the bacterial genome . have the ability to replicate indepedent of bacterial cell genome using an (ori) encodded in a dna sequence inside plasma.
what are linkers
synthetic DNA fragments containing restriction sites
an ideal cloning vector has what 3 things
an ori, one or more selectable markers, recognition sites for one or more restriction enzymes
why is selectable marker in a plasmid necesarry
because it allows cells containing the vector to be selected or identified
why are recognition sites important for a vector
plasmids need these sites for cleavage because this is where the restriction enzymes (restriction endonucleases) can cut the DNA at specific sequences. allows insertion/ removal of specific genes/ DNA fragments within the plasmid.
what happens when restriction enzymes cut at these sites
creates sticky ends (overhangs/blunt ends) that can be used to join other pieces of DNA. allowing precision when inserting new foreign DNA.
what is common when cleaving the foreign DNA and the plasmid
typically cleaved with the same restriction enzyme
what is notable about plasmids used today
they have highly engineered sequences to optimize various functions (high copy number, inducible, high protein expression).
how did fred griffiths exp demonstrate transformation in bacteria.
activated by high stress enviornment, bacterium takes up foreign DNA from dead bacterium and incorporates it within its genome, survival mechanism, provides anatiobiotic resistance
does DNA have to be bacterial in source to experience transformation
no (hence recombinant DNA)
what does it mean to make bacteria competent
make it able to take up foreign DNA
how do we co opt bacteria transformation ability
after creating a recombinant plasmid with the gene of interest, the plasmid is introduced into bacteria under special conditons allowing them to take it up. once it is taken up the bacteria can replicate it and make copies of the plasmid.
do all bacteria sucessfully take up the plasmid
no, that is why scientists use antibiotic selection when experimenting, since the plasmid generally has antiobiotic resitance, only bacteria that succesffully taken up the plasmid will survive when grown on agar plant with antiobiotic. allows for selection and isolation
what is easy expansion
bacterial cells with their resident plasmids can be quickly cheaply expanded
what is indefinite storage
these transformed bacteria can be put in glycerol and frozen and stored at -80 celsisus with little to no effect on cell health of DNA degradation (ability to store new genetic seuqences)
is circular dna stable
yes circular DNA is very stable
how do we obtain/ purify ONLY transformants
using selectable markers on the plasmid (genes that encode a marker) which are typically visual or growth selective.
what is an example of a selectable marker
ampicillin resistance gene. when bacteria are spread on a lawn of media containing ampicillin only transfromants will grow into colonies
what is then done with the transfromant colonies
a single colony is picked and cultures into large isogenic (same genotype) bacterial cultures. plasmid DNA can be purified from these cultures
what do plasmids and transformations allow
allow us to multiply and maintain DNA sequences indefinitely
what are endonucleases?
enzymes that cleave the phosphodiester bonds within a polynucleotide chain. They cut DNA or RNA at specific internal sites, unlike exonucleases, which remove nucleotides from the ends of the chains.
what are restriction enzymes
DNA endonuclease enzymes
where were restriction enzymes found
they were discovered in bacteria as a defense mechanism agaiant invading viruses by functioning as a part of a bacterial innante immune system by identifying and cutting foregin DNA. stop viral DNA from integrating into the bacterial genome
why is it extermely useful that different enzymes have different recognition sequences?
allows researchers to target and cut DNA at very specific sites. This enables precise genetic manipulation, such as cloning, gene editing, and the construction of recombinant DNA
what are the 3 ways of cutting that enzymes can do ?
5' overhang, 3' overhag, blunt end
5' overhang? 3' overhang? blunt end?
5' leaves a cut in a way that leaves a single stranded 5' end, 3' leaves a single stranded 3' end. blunt end cuts straight through both strands leaving no overhangs
what needs to happen for two dna sources to hybridize
they need to be cut/digested with same enzyme and mixed for the overhang ends (sticky ends) to hybridize due to the correct homology
what is the role of T4 DNA ligase
covalently seal (ligates) 'nicks' in sugar phosphate bonds on two ends. Thus, creates stable double stranded piece of DNA.
Where do recognition sequences occur in plasmids
Recognition sequences occur at specific locations in the plasmid where the restriction enzyme cuts. also called restriction sites and only occur at this one location in plasmid.
how are restriction enzymes named?
Each restriction enzyme is named based on the species of bacteria in which it was discovered, with the first portion of the name (in italics) representing the bacterial genus or species.
what is a common method of visualizing DNA
through gel electrophoresis
what is the gel electrophoresis medium
gels (agarose or polacrylamide) where one side has small wells
how does Gel electrophoresis work pertaining to its current
gels are covered in ionic buffer and electric field is applied, DNA is made of acids and acids are negatively charged so they slowly migrate to th eother end of the gel (postive pole)
what moves faster in the gel smaller or larger fragments
larger DNA fragments move slower through the gel matrix thus run slower than small DNA fragments (stay closer to the negative side)
how do we see the DNA after it travels through the gel
DNA is stained with soemthing that binds to the nucleic acids (ex. ethidium bromide), this can be imaged with a cemra and a an appropriate light source (ex. UV light). certian DNA bands can be cut out of the gel and purified
how do we copy and then multiply/amplify speciic DNA sequences in a very efficent manner
polymerase chain reaction (PCR)
who developed PCR
kary mullis 1983
breifly in 3 steps how do cell replicate
1. single stranded DNA template (unwound by Helicases)
2. replication machinery (DNA polymerase)
3. RNA primers (created by primase)
4. free dNTPS
briefly explain PCR. what is used to seperate DNA, how does it receieve dna polymerase, what primers are used how are dNTPs supplied.
heat used to seperate double stranded DNA instead of helicase
dna polymerase supplied a test tube
dna primers (synthetically created)- small chains of nucleotides (17-25) that are complementary to the template and provide 3'-OH end for the polymerase to work with
free dNTPs supply in a test tube
what does PCR stand for
polymerase chain reaction
what is in the PCR reaction mixture (tube) - 5
1. DNA template ( double stranded)
2. primers (forward and reverse)
3. dNTPs
4. DNA polymerase (heat resistant)
5. buffer (ions)
what is step 1 of pcr
heat to 90-100 celsisus for a few minutes
how do primers anneal to single strands in the second step of PCR
cooled 30-60 celsius rapidly for less than a minute. primers are in high conc, rapid cooling give advantage for small primer to bind template before larger single stranded DNA comes back together.
what is used to flank a sequence of interest in pcr in step 2
both forward and reverse primer
what happens during step 3 of PCR
heat to 72 celsisu, polymerase tolerates the rapid heating in step 1
how was PCR revolutinized
by the discovery of thermal resistant DNA polymerase in bacteria, thermus aquaticus
what is a thermocycler
machine that allows repeated cycling of 3 primary steps (heating and cooling) 25-35 times
in pcr what does each cycle of robust amplification do to the number of target dna
ideally doubles the number of copies of the target DNA.
copies of DNA= 2^N X inital copies of DNA. N is the number of PCR cycles.
due to specificty imparted by the forward and reverse primers, we are amplifying only a specific sequence... what does this mean?
Forward Primer: Binds to the beginning of the target sequence on the sense strand of DNA.
Reverse Primer: Binds to the end of the target sequence on the antisense strand of DNA.
By choosing primers that are complementary to specific regions flanking the target sequence, you ensure that only the desired segment of DNA is amplified
how long of primers do you want for PCR
20nt long
how should you design the forward and reverse primers
Forward Primer: This should be designed to bind just upstream of the start codon (usually in the 5' untranslated region or UTR).
Reverse Primer: This should be designed to bind just downstream of the stop codon, in the 3' UTR or exon.
what does DNA sequencing and DNA libraries allow us to do
locate a gene/DNA sequence
true or false, every cell in our bodies have the same genetic code
true
are all the genes in a genome expressed in a cell
no, on average, only 1/10th the genes in the genome is expressed in a cell
how does the same genetic code without our cells give rise to very diverse tissues, all with their own different and unique functions
cells of different tissues express only a subset of genes
what is the first step of how cDNA is created
the first step is isolation of total RNA. first you collect cells or tissues of interest than extract the total RNA from the sample.
after isolating total RNA from your sample the next step is to generate complementary DNA from mRNA... how is this done?
with oligo (dT) primers which are short sequences od deoxythumidine which bind to the poly-A tail of eukaryotic mRNA molecules. the poly A tail is unique to mRNA so the oligo primer ensure that only mRNA is reverse transcribed. (this is the starting point for the reverse transcription reaction)
what does reverse transcriptase do after the oligo (DT) primers
RT is an enzyme that synthesizes cDNA from mRNA, it synthesizes the first strand by copying the mRNA template. it had both RNA- dependent DNA polymerase activity (mRNA--> cDNA) and RNase H activty (degrade RNA template after cDNA is made)
in what direction does RT synthesize
synthesizes cDNA from the 3' end of the mRNA and works toward the 5' end. results in a single stranded cDNA copy of the mRNA. RNase nicks the mRNA strand.
where does DNA polymerase I come in when creating cDNA
after RT and the first strand of cDNA being made, DNA polymerase I which comes from E.coli and has both polymerase and exonuclease activies is used to syntehsize the second strand of cDNA using dNTPS. adding complementary bases to the first cDNA strand.
what else does DNA polymerase do after synthesizing the second strand
since it also has RNase H activty, which removes the RNA strand from the original template, so it can be degraded after reverse transcription
what is the end product after oligo, RT and DNA poly I
double stranded cDNA molecules where one strand is complementary to the mRNA and the other strand is the reverse complement
what happens after you have the double stranded cDNA
it can be inserted into a cloning vector.
how is the cDNA cloning vector prepared
you will cut the vector (plasmid) with appropriate restriction enzymes to generate sticky or blunt ends that will allow for insertion of the cDNA.
whar happens after the vector for cDNA is prepared
the double stranded cDNA is ligated into the vector using DNA ligase which forms covalent bonds between the cDNA insert and the vector. now the cDNA is part of the plasmid vector and can be introduced into bacterial cells
what is transformation (cDNA)
how the ligated is introduced into competent E.coli, the bacteria that actually take up the plasmid will begin to replicate creating clones of the cDNA insert. the colonies can then be screened to identify the ones containing the desired cDNA insert
what is specific about cDNA library
it is specific to a particular tissue, cell type, state or time. they can be created to capture gene expression profile of specific conditon or stage in the life cycle of an organism
why do we use DNA sequencing
helps us understand the protein sequence a gene product will make (gene structure/function) as well as for designing primers for downstream molecular cloning of these gene sequences into plasmids
who was sanger sequeuncing made by and what is also called
sanger sequencing is the standard used for seuqencing DNA fragments in laboratories. created by frederick sanger and colleagues in 1970s, it is also called dideoxy sequencing.
how is dideoxy (sanger) sequencing similar to PCR (3)
1. DNA replication with DNA polymerase
2. DNA template
3. primers
what are the 5 differences between PCR and sanger sequencing
1. number of primers
2. use of ddNTPS
3. concentration of ddNTPS
4.labeling for detection
5. amount of DNA required
pcr vs ss: number of primers
pcr- 2 primers (F and R)
ss- 1 primer
pcr vs ss: use of ddNTPS
PCR- uses dNTPS (deoxyribonucleotide triphosphate) to extend the DNA strand
ss: uses ddNTPS (dideoxyribonucleotide triphosphates), which lack the 3' hydroxyl group, causing DNA synthesis to terminate when incorportaed.
pcr vs ss: concentration of ddNTPS
PCR: dNTPS are present in equal amounts
ss: ddNTPS are present at a 100 fold lower conc than dNTPS to ensure controlled termination of DNA synthesis
pcr vs ss: labelling for detection
pcr: no special labelling is required
ss: primer or ddNTPS are labelled (radioactive or fluorescent) to visualize and read the sequence after electrophoresis
pcr vs ss: amount of DNA required
pcr: small amount of template DNA as the target region gets amplified
ss: large amount of DNA becuase PCR amplification or cloning comes before sequencing to produce a large amount of target DNA
which version of ss labels the primer
the original version the primer is radioactively on the 5' end. the automated version the primer is not labelled
what is labelled in the automated version of ss
each ddNTP (A,C,G,T) is labelled with a different fluorescent probe (illustrated by green, purple, black, red etc.)
what is the difference in tube # in the automated and original version of ss
original: many copies of target DNA and primer are added to 4 tubes
automated: everything goes into one tube and one well on gel
in the original version of ss what goes in each tube (4)
1. lots of dNTPS
2.DNA polymerase
3. a small amount (1/100) of one of the 4 ddNTPS (A,C,G,T)- low conc to ensure random incorporation/chain termination at various points
how are dna bands read in the orignal ss and whats it con
gel electrophoresis to seperate by DNA length, very laborious, expensive
how does automated ss read dna sequences
carried out by automated machines using laser scanners, imagers and software to read out DNA sequence
what is insulin
a peptide hormone, used in the treatment of diabetes
what must we do for to use these techniques to produce insulin (3)
1.identify human tissues that expresses insluin (pancreas)
2. purify RNA from pancreatic tissue
3. use purified RNA to create cDNA from pancreatic mRNA
after making the insulin cDNA what needs to be done
using known genetic sequence of insulin,design a forward (3' end) and reverse (complentary 5' end) DNA primer for inulin cDNA
then to the 5' ends of the forward and reverse primer add an extenstion of a few nucleotides which contain a restriction enzyme recognition site
what do the primers do for the insulin cDNA
in a PCR reaction they amplify the insulin cDNA
after insulin cDNA is amplified then what happens
- load you PCR product on an agarose gel, then use gel electrophoresis to make sure your DNA band is the size you expect
after bands are made from the insulin DNA then what happens
cut your insulin cDNA band out of the gel and purify the DNA from the agarose gel
after insulin cDNA band is cut out and purified then what should you do
add restriction enzyme to your DNA fragment to cut at the restriction sites that you put on the ends, use the same enzyme to digest a plasmid optimized for expressing a protein in E.coli
why do we use the same RE for the dna fragment cutting and to digest the plasmid? what happens after this step?
we use the same RE to digest both becuase it will leave complentary DNA ends that will fit together. then ligate the insulin cDNA into the expression plasmid
what happens with the newly synthesized plasmid with the insulin gene
it gets transformed into competent e.coli cells, then we allow it to grow then spread on a culture plate to obtain single colony isolates
after the insulin colonies grow then what do we do
we perform a PCR on anumber of different bacterial colonies (1-4) to confirm they contain the insert and not just a religated plasmid
what do we have to not just have a religated plasmid
designing one primer in the plasmid and one primer within the insulin cDNA would produce a product
how do we know we have a postive PCR product
use gel electrophoresis to visualize wheter you have a postive PCR product or not
after we get our positve PCR product results what do we do
After selecting colonies or cultures with the plasmid containing the insert, we purify the plasmid DNA. We then design primers that span both the insulin cDNA and the surrounding plasmid regions (flanking parts). The plasmid is sent for sequencing, and the resulting DNA sequence is analyzed to confirm the correct insertion of the insulin cDNA and ensure there are no mutations.