Maria, Rachel, Pooja and Rami - MCB 720, HW 2 - 02/09/2011
Question 1:
cDNA library construction:
Isolate total mRNA from the beaver salivary glands. Using enzyme reverse transcriptase, we can
transcribe the mRNA to cDNA. Remove the RNA strand with alkali and add poly G tails using terminal
transferase. Hybridize the single stranded cDNA with oligo-dC primer. A complementary strand is
synthesized using enzyme DNA Pol. DNA ligase is used to fill in the gaps. The cDNA strands synthesized
can be protected from methylation (using methylase), so that they are not degraded when expressed in
a vector or a plasmid. Short dsDNA molecules (linkers) that contain the recognition site for a particular
restriction enzyme are ligated to the ends of cDNA using DNA ligase. Following this, treatment with a
particular restriction enzyme like EcoRI will produce cDNA molecules with sticky ends.
In a separate procedure, a vector or a plasmid is treated with the same restriction enzyme to produce
sticky ends that are complementary to the sticky ends produced in the cDNA. After incubation of this
cDNA with DNA ligase and the vector containing the sticky ends, some of the vectors will take up the
cDNA fragments. The vector can then be used to infect cells like E.coli for cloning.
Method 1: Using the amino acid sequence of Beaver Salivary Protein:
This method is based on the already known amino acid sequence of a trypsin-digested peptide from
Beaver Salivary Protein (BSP). The goal is to design an oligonucleotide probe to identify the cDNA clone
of interest encoding the protein BSP. With the already known partial BSP sequence, we can obtain its
mRNA sequence, using a codon table available for different species. These probes can be radiolabeled
using 32P-dNTP and polynucleotide kinase. Grow the beaver genomic library containing cells onto a plate
and lift some of these using a nitrocellulose membrane (NCM). Incubate this NCM with previously
radiolabeled degenerate probe, which will hybridize with the clone of interest. After detecting the clone
of interest using autoradiography, we can go back to the plate and collect the matching colony (which
potentially contains the beaver salivary cDNA clone).
Method 2: Using an antibody against Beaver Salivary Protein:
This method makes use of the antibodies generated in rabbits against the BSP protein, as well as the use
of expression libraries. These antibodies could be used as probes to immunologically find the resulting
fusion proteins. Beaver cDNA library could be cloned near the restriction site of bacteria containing a galactosidase gene close to a promoter. This newly inserted cDNA forms a fusion protein as it expresses
-galactosidase along with BSP. After plating the cells containing these expression libraries, we can lift
them onto a NCM. Incubate the NCM with the rabbit anti-beaver salivary protein. Only the protein of
interest will bind to this specific antibody. Following this, add secondary antibody conjugated to alkaline
phophatase or horseradish peroxidase. Using a substrate, we can identify the colonies expressing the
protein of interest. After identifying the BSP containing colonies, we can go back to the plate and collect
the matching colony (which potentially contains the beaver salivary cDNA clone).
Method 3: Differential screening
This method is based on isolating cDNA that is expressed in one situation and not in another. In this
experiment we are looking at two cases: (1) When the BSP is induced by consumption of wood and (2)
When BSP is not expressed (no consumption of wood).
Beaver Saliva is cultured in the presence (+) and absence (-) of wood shavings/wood homogenate. The
cells will express some mRNA in common, but the beaver salivary mRNA will not be present in the cells
cultured without the wood homogenate. Isolate the mRNA and reverse transcribe it using the reverse
transcriptase method. Label the mRNA from both (+) and (-) cells with 32P-dNTP. These labeled probes
can be used later. Grow the beaver cDNA library containing cells onto a plate (with or without wood
shavings/homogenate) and then lift the cells using two NCM’s from the same plate of cells. Hybridize
one NCM with the radioactively labeled mRNAs obtained from the (+) plate and the other obtained from
the (-) plate. Expose the NCM using autoradiography and then compare the (+) and (-) lifts. Any cDNA
clone that hybridizes the lifts from the cDNA library and not the other is the one that expresses the BS
cDNA. After identifying the beaver salivary CDNA containing colonies, we can go back to the plate and
collect the matching colony. We can repeatedly screen this isolated cDNA clone, till we get
approximately 100% of the cell population expressing BSP.
Verification of the cDNA clone of interest
Northern blotting: Isolate the total RNA from the beaver salivary cDNA clone containing cells that
express the protein of interest. Samples are then separated according to size on a gel by electrophoresis.
After transferring to a NCM, we can add a labeled DNA or RNA as a probe. The RNA of interest can be
detected by the presence of a band after exposing the blot using autoradiography.
Hybrid select translation: DNA from the beaver salivary cDNA clone containing cells was extracted,
denatured and immobilized on a NCM. This NCM is soaked in total mRNA obtained from the beaver
salivary glands. The NCM will bind to only one specific mRNA. After washing all the unbound mRNA,
elute the bound mRNA for in vitro mRNA translation. After mRNA translation the protein can be
detected by immunoprecipitation or electrophoresis.
DNA sequencing: DNA sequencing is the determination of the precise sequence of nucleotides in a DNA
sample. The dideoxy method or Sanger method is the most popular method of DNA sequencing. The
main steps of DNA sequencing are: 1) Obtaining DNA from the cell 2) Performing a sequencing reaction,
which requires a primer and DNA polymerase. The primers or nucleotides included in these reactions
contain different fluorescent labels 3) Separating DNA by size using capillary electrophoresis 4)
sequencing the DNA by a specialized software.
Question 2:
Explain how to construct a bacteriophage genomic library and isolate a genomic clone for BSP using
the previously obtained cDNA clone:
First, genomic beaver DNA will be partially digested with Sau3A (cleaving the DNA into fragments of
~20kb). A bacteriophage can then be cut with BamHI, forming sticky ends, which are complementary to
the sticky ends produced by Sau3A. Thus, upon incubation of the digested genomic DNA with the cut
bacteriophage and DNA ligase, some bacteriophages will take up the genomic DNA fragments. The
bacteriophage will package the DNA into its “head” component, after which the tail of the phage will
attach, forming a completely assembled bacteriophage. Then the bacteriophages can be combined with
bacterial cells (which they will infect), and the bacteria can be grown on antibiotic-resistant media
(depending on the type of resistance the phage carries). The bactriophages will replicate in the bacteria,
eventually lysing the cells, and therefor “displaying” the various genomic DNA segments on the plate.
Plaques (clones from one bacterial cell) are formed, and these can be blotted onto nitrocellulose, which
binds permanently in ssDNA. The previously prepared cDNA clones can be labeled and used as probes
for the genomic DNA, thus allowing for the isolation of the genomic clone.
How many bacteriophages must be screened to find the BSP gene with a 98% degree of certainty?
What about using cosmids, BACs, YACS, cosmids?
The number of bacteriophages that will need to be screened to find the gene of interest is given by the
following formula: N = ln(1-P)/ln(1-f), where N = number to be screened, P = probability of locating the
gene of interest, and f = average size of genomic insert/entire genome size.
Therefore:
- If using a bacteriophage (which allows for an insert size of 20kb):
N = ln(1-0.98)/ln(1-(20kb/3 x 106 kb)) = 5.87 x 105 candidates
- If using a cosmid (45 kb insert maximum):
N = ln(0.02)/ln(1-(45 kb/ 3 x 106 kb)) = 2.61 x 105 candidates
- If using a BAC (300 kb insert limit):
N = ln(0.02)/ln(1-(300 kb/ 3 x 106 kb)) = 3.91 x 104 candidates
- If using YAC (1000 kb insert possible)
N = ln(0.02)/ln(1-(1000 kb/ 3 x 106 kb)) = 1.17 x 104 candidates
What sequence information will be present in a complete BSP gene clone, but absent in a full length
BSP cDNA clone?
The BSP genomic clone will contain all of the information encoded by the BSP gene, including exons and
introns. In contrast, the cDNA clone will not contain any introns, since it was reverse transcribed from
mature (spliced) mRNA.
Question 3:
Restriction map of the genomic DNA
Each segment represents 1 kb
MCB 720 Homework (Jia, Alamzeb, Salah, Yongli )
Question1
In order to separate the cDNA clone that encodes this specific protein, three ways:
1. Use the immunological screening method for the cDNA library with a primary antibody and a labeled secondary
antibody.
First, we need to obtain the cDNA library: Obtain mRNA from saliver cells or the salivary gland cells in the beaver.
(run cell extract on a column with oligo-dTs matrix); mRNA hybridize with oligo-dT primer; Transcribe mRNA in to
cDNA (Reverse transcriptase); Remove RNA with alkali, add poly(dG) tail; Hybridize with oligo-dC primer;
Synthesize complementary strand; Protect cDNA by methylation; Ligate cDNA to linkers; Cleave with EcoRI; Ligate
to λ arms, and package in vitro. Infect E. coli; Do the bacteriophage λ cloning system.
So we get individual λ cDNA library. Then we need to separate the cDNA clone we want, that encodes the BSP.
Since the antibody has already been made from rabbit (primary antibody), the secondary antibody can be made
according to the primary antibody by injecting the rabbit antibody into sheep. Label the secondary antibody with
125
I. Then we made protein from the cDNA. With the help of lac promoter, we clone the cDNA into bacteria cells,
by using in vitro packaging plate on bacterial lawn. Then overlay a nitrocellulose filter, 30s, remove the filter, the
proteins would bind to the nitrocellulous. Incubate filter with primary antibody, wash, incubate with radiolabeled
secondary antibody. Then we perform autoradiography, on the X-ray film, the labeled one would be the cDNA
clone we want. Pick them up on the original cDNA plate at that position using a pipette tips, then clone them, do
the procedures again and again, until the protein we get would be 100% BSP.
This way can also be used to detect if the cDNA clones we made code for BSP.
2. Use the oligonucleotide probe based on the known amino acid sequence (since the BSP is already purified and
a part sequence got by using trypsin is “SLFDFADKNRGSYSGSCPFYCSYSGYQDELLWAAAWLYK”).
Choose about 7 amino acids, which encodes for the least-degenerate 20-base region. Then prepare 20mer
degenerate probe (labeled with -32P) to screen the cDNA library. Lay the nitrocellulous filter on the plate where
our cDNA library is, then incubate filter in alkaline solution to lyse phages and denature released cDNA, making
ssDNA bound to filter. Hybridize with labeled probe, then perform autoradiography. Signal appears over phage
DNA that is complementary to the probe, which is the cDNA clone we want. Pick the spot on the cDNA library
plate, clone them again, then repeat the procedures, until the cDNA we get can 100% percent bind to the labeled
probe. Then purify it, and we get the purified cDNA clone.
3. Plus/minus or differential screening. The BSP cells might be heat shock or have other properties, which we
need to test. But here we use the serum. Perform two experiments together, one treat with serum, the other not,
get we get serum induced mRNA and the one that is not serum induced. Both do reverse transcription with 32P
labeled dNTPs. Use these as probes for ssDNA we get from the cDNA library on 2 separate nitrocellulous filter. By
antoradiography, we get the isolate cDNA clone from phage on the master plate.
4.
c-DNA clone separation from Expression Library
To obtain a cDNA encoding BSP protein, constructing expression library is another method. First, an m-RNA is
obtained from cells that encode BSP protein, and then expression libraries are prepared by using plasmids. As one
residue could be encoded by different codons, special plasmids are used for cloning each cDNA. Along a strong
promoter, the vector contains another gene for expressing another protein. Then, the expression library plasmids
are transferred to cell culture that doesn’t encode BSP. Both proteins are expressed and a fusion protein is
obtained from the cDNA and the gene already in the plasmid. Then the cells are detected for the specific protein
by using an antibody. The detected cells are then separated and the cDNA that encodes BSP can be isolated from
the cells.
Three experiments to verify the protein:
1. Northern blotting is one of the methods to characterize a clone gene. A transcribed m-RNA can be detected
by using this technique. The denatured and individual RNAs are separated by gel electrophoresis according to
size and transferred to a nitrocellulose filter. The attached mRNA with the nitrocellulose filter is exposed to a
labeled DNA probe, followed by autoradiography.
2.
3.
Southern blotting is another technique for detecting the cDNA sequence of a protein. This procedure can
be applied to a millions of c-DNA fragments, and the labeled probe that hybridized to the c-DNA of
interest will provide proof on the autoradiogram.
Western blotting, ………………
Answer to Q2. (A). 1- We isolate the DNA from the beaver and use partial digestion with Sau3A (GATC
restriction site) into 20 kb fragments with sticky ends.
2- The DNA bacteriophage λ will be cut with BamHI (GGATCC restriction site) to remove the replaceable
region and obtain sticky ends.
3- To avoid relegation of the vector, we need to treat λ vector with alkaline phosphatase (SAP) to
dephosphorylate the ends of the vector.
4- We eliminate the SAP buffer and mix the vector with DNA fragments in presence of T4 DNA Ligase
which will allow the ligation of the fragments randomly to the λ vectors.
5- The recombinant product is packaged with in vitro phage-assembly system resulting a recombinant λ
virion containing the beaver fragments.
6- The λ phages will be placed into E. coli and grown on selective media resulting λ phage plaques on E.
coli lawn.
7- We place a nitrocellulose filter on the plate to pick up phages from each plaque.
8- We incubate nitrocellulose filter into alkaline solution to lyse phages and denature released DNA.
9- To identify similar DNA fragments to the cDNA clone of BSP, we will use our clone as a homologous
probe labeled with 32P (random primer labeling) to perform an autoradiography. Therefore, we can
identify the clones encoding for BSP protein.
(B). How many bacteriophages (N)?
The beaver genome is 3 x 109 bp.
The insert capacity of bacteriophage λ is 10-20 kb.
N = ln (1 - 0.98) / ln (1 – 10kb/3 x 109) = 1.17 x 106
N = ln (1 - 0.98) / ln (1 – 20kb/3 x 109) = 5.86 x 106
(C). Cosmid vector: (35-45) kb
N = ln (1 - 0.98) / ln (1 – 35kb/3 x 109) = 3.35 x 105
N = ln (1 - 0.98) / ln (1 – 45kb/3 x 109) = 2.61 x 105
BAC vector: (50-300) kb
N = ln (1 - 0.98) / ln (1 – 50kb/3 x 109) = 2.35 x 105
N = ln (1 - 0.98) / ln (1 – 300kb/3 x 109) = 3.91 x 104
YAC vector: (100-2,000) kb
N = ln (1 - 0.98) / ln (1 – 100kb/3 x 109) = 1.17 x 105
N = ln (1 - 0.98) / ln (1 – 2000kb/3 x 109) = 5.87 x 103
Increasing the capacity of the vector, the number of clones will decrease.
It will be difficult to sequence large fragments.
(D). The sequence that will be absent from the full length BSP gene clone will be the intron.
Q3
Alastair, Adam, Craig and Xaio
1) The BSP cDNA library would be screened using a protein-specific oligonucleotide probe, an antibody
to the BSP, or positive-negative screening. First, however, a cDNA library would be constructed. Salivary
gland total RNA is isolated from a beaver after BSP has been induced by wood consumption. Then,
affinity chromatography is performed using oligo(dT)-cellulose, so mRNAs with poly A tail can bind to
the matrix. The column is washed with NaCl solution and the mRNAs eluted with water. Eluted mRNAs
are hybridized with an oligo-dT primer in the presence of reverse transcriptase and dNTPs to synthesize
the first-strand cDNA. The RNA-DNA hybrid is then treated with an alkali, which degrades RNA, leaving
only the cDNA. A 3' poly G tail is added using terminal transferase. The complementary cDNA strand is
then synthesized by DNA polymerase using an oligo-dC primer. The double-stranded cDNA is treated
with EcoRI methylase to prevent EcoRI restriction. Linkers containing EcoRI sites are ligated to the
cDNA flanks by DNA ligase. Multiple linkers could attach to the cDNA but EcoRI restriction leaves only
one at each end. The cDNA with EcoRI sticky ends is ready for ligation to λ arms for phage packaging in
vitro. Individual λ cDNA clones can grow on a bacterial lawn, showing up as plaques.
An oligonucleotide probe can be designed from the amino acid sequence of the trypsin-generated
peptide. The specific DNA sequence encoding the fragment is unknown so an oligonucleotide probe is
made from the least degenerate region of about 20bp. The desired oligonucleotide is synthesized with a 5'
hydroxyl group so that γ32P phosphate from radio-labelled ATP can be added to it. This process, called N
labelling, allows detection of the probe through autoradiography.
The bacterial colonies or phage plaques on the cDNA library plates are blotted with a nitrocellulose
membrane. The membrane is treated with sodium hydroxide to lyse the cells and render the target DNA
single-stranded. After incubation with the probe, the membrane is washed to remove any unbound probe.
The probe can then be visualized by incubating the membrane with photographic paper. The image
produced can be used to identify the original colonies or plaques on the library plates that contain the
DNA fragment of interest. Colonies should be restreaked and the blotting process repeated to ensure that
the correct colonies have been selected.
For antibody screening, cDNAs must be inserted into an expression vector, downstream of a strong
constitutive promoter, so that it is transcribed and translated into a polypeptide for which we have an
antibody. cDNA library plates are blotted with a nitrocellulose membrane, which is treated with sodium
hydroxide to release proteins from the attached cells. The membrane is incubated with a primary antibody
specific to the peptide of interest, such as the previously-obtained rabbit BSP antibody. Unbound antibody
is washed off and a secondary antibody that is conjugated to a reporter such as the alkaline phosphatase
enzyme is applied. This secondary antibody is specific to the primary antibody. This labels
colonies/plaques that have bound the primary antibody, and amplifies this signal. We then assay for
reporter activity or radioactivity to identify clones of interest.
BSP is induced by wood consumption, therefore we can use positive-negative selection to identify it and
other induced proteins. mRNA is isolated from the salivary glands of a beaver that has been fed wood,
then 32P-labelled cDNA probes are made using the N labeling method. Secondly, a cDNA library is
constructed. Thirdly, mRNA is isolated from the salivary glands of a beaver that has not been fed with
wood. Two sets of cDNA probes with 32P labeling are then produced from the two mRNA extracts. The
library is blotted with two different nitrocellulose membranes and each membrane is hybridized with a
different set of probes. After autoradiography, plaques absent from plate that has been hybridized with
non-induced probe set and present in the one with wood-induced probes contain cDNA from a woodinduced mRNA
In northern blotting, total RNA isolated from the salivary glands of a beaver after wood consumption is
separated by gel electrophoresis and then blotted onto nitrocellulose. A labeled DNA probe is made from
the cDNA clones of interest. The probe should bind to one of the electrophoresed mRNAs. This is visible
on the X-ray film after autoradiography. We can identify the corresponding RNA band in the gel, purify
the mRNA, make cDNA, and clone that cDNA into an expression vector. Finally, we can detect if the
protein translated is our target protein. In the hybrid-select translation, cloned cDNA can hybridize with
cellular mRNA to bind the specific mRNA, which is then translated in vitro. This protein is compared to
the previously-purified BSP to verify that the cDNA clone codes for the BSP protein. In the DNA
sequencing method, we can sequence the cDNA clone then compare its DNA sequence with the putative
DNA sequence that encodes the BSP that we previously purified. From this comparison, we can see if the
cDNA clone indeed codes for the BSP protein.
2) A) For creating a genomic library for the beaver you need to start by extracting genomic DNA from
any part of the beaver. The next step is to cut the genomic DNA with a restriction enzyme, but not a
complete digest, only a partial digest so you get fragments around 20kb so that they fit into
bacteriophage lambda. Once the DNA is cut you need to remove 5’ phosphate groups so the fragments
don’t religate to themselves, the enzyme that does this is alkaline phosphatase. Then you must open up
the phage with the same restriction enzyme that you used to cut the DNA and then ligate the DNA
fragments into the phages with T4 DNA ligase. Now that you have a bunch of bacteriophage lambda
clones you need to infect a lawn of E. coli with the bacteriophages. The E. coli will replicate the
bacteriophage then, the E. coli will lyse releasing the contents of the phage onto the medium creating a
plaque which is representative of a single bacteriophage with a single DNA fragment. The next step is to
bind the phage DNA to a nitrocellulose membrane and treat it with a base to denature the DNA to single
stranded. To screen the library you would need to make a probe that is complementary to the gene of
interest that has some sort of label that would be observable. To create the probe I would use the
random primer method for creating a radiolabled probe. To do this I would use the DNA of the beaver
salivary gland protein to prime off of, to do this I would denature the DNA in the presence of random
primers. One of the dNTP’s would be radiolabeled on the alpha phosphate and use the klenow
fragment of DNA polymerase to amplify the probe that will incorporate radiolabels. The next step is to
wash the probe over the nitrocellulose membrane and it will bind to a complementary sequence and an
autoradiograph can be viewed to identify the bacteriophage with the gene of interest. You could go
back to the original plate to isolate the DNA from the phage.
B) N= ln(1-P)/ ln(1-F)
N= ln(1-.98)/ln(1-20,000bp/3,000,000,000bp)
N= -3.91262/-0.000006667
N= 586,510 bacteriophages
C) The use of vectors with larger insert capacity would increase the amount of DNA tht could be fit into
the vector and also reduce the amount of time that you would have to do the initial restriction digest.
By increasing the capacity of the vector you also reduce the amount of clones that need to be screened
to identify your gene.
1) N= ln(1-P)/ ln(1-F)
N= ln(1-.98)/ln(1-45,000bp/3,000,000,000bp)
N= -3.91262/-0.000015
N= 260,802 cosmids
2) N= ln(1-P)/ ln(1-F)
N= ln(1-.98)/ln(1-300,000bp/3,000,000,000bp)
N= -3.91262/-0.0001
N= 39,120 BAC’s
3) N= ln(1-P)/ ln(1-F)
N= ln(1-.98)/ln(1-2,000,000bp/3,000,000,000bp)
N= -3.91262/-0.000668
N= 5867 YAC’s
D) Information that will be in a gene clone and not a cDNA clone will be regulatory information like
promoters and enhancers. Also it will contain intron sequences.
Question 3
The following map satisfies all the above fragment lengths and places the southern blot
hybridized fragments in the same vicinity within each digest. Note that EMBL3 DNA is found
from positions 0kb – 20kb and 35kb – 44kb. Also note that restriction sites for all three enzymes
(EcoR1, BamH1, and Sal1) must exist at 20kb and 35kb to allow for 20kb and 9kb fragments in
all digests.
MCB 720 Homework/Practice Exam Answers (key points to mention in your answers)
1. A. 3 screening strategies for the BSP cDNA:
Strategy 1: Screen a cDNA library with an oligonucleotide probe designed based upon the least
degenerate region of BSP peptide sequence; label with -32P-ATP and polynucleotide kinase.
Specifically,make an oligonucleotide corresponding to the least degenerate region (i.e.,
FDFADKN; thus, 5’-TTT/C GAT/C TTT/C GCX GAT/C AAA/G AA-3’ where X=G,A,T,C)
Strategy 2: Screen a cDNA expression library with the BSP antibody
Strategy 3: Screen a cDNA library made from the salivary gland mRNA of beavers allowed to
consume wood using (radio)labeled beaver salivary gland mRNA (or first strand cDNA) from
wood-fed and non-wood-fed (control) beavers [i.e., differential or plus/minus screening]
B. For details of cDNA synthesis and cloning see lecture notes & book (the names for
enzymes used here should be included in your answer).
C. Verification:
1. Perform DNA sequencing of your cDNA sequence and look for sequences corresponding to
(i.e., matching) the known BSP peptide sequence
2. Use hybrid select translation with 35S-Met and see whether a radiolabeled 55 kDa protein is
observed upon SDS-PAGE (note that this can also be done in combination with
immunoprecipitation with the BSP antibody prior to running SDS-PAGE)
3. Probe northern (RNA) blots of salivary gland RNA from wood-fed and non-wood-fed beavers
with your radiolabeled cDNA and determine whether you see an appropriate size RNA (i.e,
[55,000 daltons x 1 AA/100 daltons x 3 nt/AA] + 500 nt = ~2,150 nt RNA) that is induced by
wood feeding.
Other: express your cDNA and hope the antibody recognizes it (a problem arises if dealing with
a partial length cDNA since it may not encode the epitope region recognized by the antibody)
2. See lecture notes and book for genomic library construction in a bacteriophage vector (partial
restriction enzyme digestion is key). Library screening involves hybridization of nitrocellulose
plaque lifts to your BSP cDNA probe produced by random primer labeling or nick-translation
using an -32P-dNTP.
N=ln (1-P)/1n (1-f); note beaver genome size is estimated to be approximately 3 billon bp
(similar to rabbits) = 3,000,000,000 bp
For bacteriophage: N=ln (1-0.98)/ln (1-20,000 bp/3,000,000,000 bp)=~586,802 clones
The larger the average size of the cloned insert, the fewer # of clones to be screened (see below):
For cosmid: N=ln (1-0.98)/ln (1-45,000 bp/3,000,000,000 bp)= ~260,800 clones
Need to screen colonies not plaques
For BAC: N=ln (1-0.98)/ln (1-300,000 bp/3,000,000,000 bp)= ~39,18 clones
Need to screen colonies not plaques, may undergo recombination events
For YAC: N=ln (1-0.98)/ln (1-1,000,000 bp/3,000,000,000 bp)= ~11,734 clones
Need to screen colonies not plaques, may undergo recombination events
-5’flanking, enhancers, silencers, promoter (CAAT and TATA), introns, 3’ flanking
3.
S
B
E
S
B
E
S
B
E
S
S
B
E