Bio Tech Exam 2 (copy; not official)
Copy number
# of plasmids in the cells (which are small)
Colony hybridization process involves
- Bacteria with recombinant DNA grow on agar plate - Nylon or nitrocellulose filter is placed over the plate -Treat filter with alkaine solution to lyse the cells and denature the DNA -bake filter or UV exposure - Denatured DNA binds to filter as single stranded DNA - Filter is incubated with a probe that is tagged with a radioactive nucleotide or fluorescent dye - Probe binds by hydrogen bonding to complementary sequences on the filter = hybridization
What are the procedures of Reverse Transcription PCR
- Isolate mRNA and use Reverse Transcriptase to make double stranded cDNA • Use PCR to amplify region of cDNA with set of primers specific for gene of interest • Run agarose gel to separate amplified cDNA fragments • Determine expression in the tissues you're comparing. • **Amount of cDNA produced in RT PCR reaction for gene of interest reflects amount of mRNA and level of gene expression.
The advantages of electroporation
- It's efficient -Fast -Requires less cells -Can introduce DNA to other types of cells
What are the steps of Northern blot
- RNA is isolated from a tissue of interest o RNA is separated by gel electrophoresis and blotted onto a nylon membrane, and hybridized to a labeled DNA probe. o Exposed bands on autoradiograph show presence of mRNA for the gene of interest as well as the size of mRNA o Can compare and quantify amounts of mRNA present in different tissues
Factors of Cloning Vector
- small enough to separated from chromosomal DNA -have an origin replication -have a copy number - multiple cloning site - selectable marker genes - RNA polymerase promoter sequences - & DNA sequencing primer flanks @ the end of multiple cloning site
disadvantage of cDNA library
-Can be difficult to make the cDNA library if a source tissue with an abundant amount of mRNA for the gene is not available
Disadvantage of genomic libraries
-Introns & Exons are cloned -Majority of genomic DNA is introns in eukaryotes so majority will have non coding pieces of DNA - Many organisms have a large genome - It's time consuming
NGS (2ndGS) with pyrosequencing
-Roche 454 commercial system • Utilizes pyrophosphate • Chemiluminescent (light releasing) reactions • ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphosulfate. • ATP is a substrate for conversion of luciferin to oxyluciferin that generates visible light.
Advantage of cDNA library
-collection of actively expressed genes in cells / tissues from which the mRNA was isolated -Introns are NOT cloned - Can be created & screened to isolate genes that tissue with an abundant amount of mRNA for the gene is not available
How does the (modern)Sangar Method work?
-ddNTPs are each labeled with a different fluorescent dye - Samples are separated on a single-lane capillary gel that is scanned with a laser beam -Laser stimulates fluorescent dye on each DNA fragment which emits a different wavelength of light for each different colored ddNTP -Emitted light is collected by a detector that amplifies and feeds this info to a computer that can run multiple capillary gels at one time = 900 bp sequence -Computer converts light patterns to reveal the sequence.
Complementary(cDNA) libraries involve
-mRNA being isolated from a tissue -making a double stranded DNA from mRNA
PCR applications are known to
-making DNA probes - studying gene expression -detection of viral and bacteria infections -diagnosis of genetic conditions -detection of trace amounts of DNA from the tissue found at the crime scene -detection of DNA from fossilized tissue
Plasmid Dna are
-used as vectors, =extrachromosomal DNA due to the cytoplasm & bacteria inside of it, -about the size of 1 to 4 kb, -& able to replicate chromosomes independently
Genomic Libraries involve
-vectors are digested with the same enzyme -DNA ligase that are used to ligate genomic DNA fragments and vectors DNA -Recombinant vectors are used to transform bacteria, & theoretically each bacteria will contain a single recombinant plasmid
Which colonies (CFUs) have the inserted gene? A. 2, 4, 5 B. 1, 3, 6 C. 1 & 2 D. All of the colonies E. None of the colonies
1, 3, 6
How to make a double stranded DNA from mRNA?
1. Add Short linker double stranded DNA sequences that have restricted enzymes to the end of cDNA 2. Cut a vector with a restriction enzyme that causes ligate fragments to create recombinant vectors 3. Then Transform bacteria with recombinant vectors
What are the steps of Calcium Choride Transformation of Bacterial Cells
1. Chill bacteria with salt for 30 mins 2. Add plasmid to DNA cells to chill on ice 3. Heat the cells & mixture for about 30 sec @ 42 degrees Celcius 4. Plasmid DNA enters bacterial cells, gets replicated, & express their genes
What are the steps of Southern blotting
1. Digest chromosomal DNA into small fragments with restriction enzymes 2. Fragments are separated by agarose gel electrophoresis 3. Gel is treated with alkaline solution to denature the DNA 4. Fragments are transferred onto a nylon or nitrocellulose filter called blotting 5. Filter (blot) is baked or exposed to UV light to permanently attach the DNA 6. Filter (blot) is incubated with a labeled probe and exposed to film by autoradiography or digital camera 7. Number of bands on film represents gene copy number.
The Steps of Colony Hybridization
1. Filter is washed to remove excess unbound probe 2. Filter exposed to X-Ray film or digital camera to detect fluorescent probe 3. Film or picture is then compared to the original agar plate to identify which colonies contained recombinant plasmid with the gene of interest.
polymerase chain reaction process
1.Target DNA to be amplified is added to a tube, mixed with nucleotides (dATP, dCTP, dGTP, dTTP), buffer with MgCl2, and DNA polymerase 2. Paired set of forwarding and Reverse Primers are added short single-stranded DNA oligonucleotides (18-22 nucleotides long) 3. Reaction tube is placed in an instrument called a thermocycler. 4. PCR cycle 5. At the end of one cycle, the amount of DNA has doubled 6. Cycles are repeated 20-30 times.
Assume you want to do 22 PCR cycles to amplify inserted DNA, how many copies will you have?
2^22 = 4,194,304
How to calculate the number of copies of target DNA starting with 1 molecule of DNA
2^n
Polymerase Chain Reaction
A technique for making copies, or amplifying, a specific sequence of DNA in a short period of time
Selectable marker genes
Allow to select for transformed colonies
Real time or quantitative (qPCR)
Can quantify amplification reactions as they occur in real time • Need special thermal cyclers that use a laser to scan a beam of light through the top or bottom of each PCR reaction. • Each reaction tube contains EITHER a fluorescent dye containing probe with quencher, or DNA binding dye that emits fluorescent light when illuminated by the laser • Light emitted via fluorescence correlates with amount of PCR product amplified • Light is capture by the detector which relays info. To the computer to provide readout on amount of fluorescence • Readout is plotted and analyzed to quantitate the number of PCR products produced after each cycle.
Fluorescene in situ hybridization (FISH)
Chromosome location of gene and copy number that can identify which chromosome contains a gene of interest
Procedure of Fluorescene in situ hybridization
Chromosomes are isolated from cells and spread out on glass microscope slide o DNA or RNA probe for gene of interest is labeled with fluorescent dye and incubated with slides o Probe will hybridize with complementary seque3nces on chromosomes on slide o Slide is washed and then exposed to fluorescent light o Wherever probe has bound to the chromosomes, it is illuminated to indicate the presence of the probe binding o Do karyotype to determine which chromosome shows fluorescence o To determine which chromosomes are homologues they are aligned according to their length and fluorescent patterns of their chromatids and use to create a karyotype
DNA libraries
Collections of cloned DNA fragments from a particular organism contained within bacteria or viruses as the host
Extension (PCR)
DNA Pol copies target DNA at 70 to 75° C
Blue White selection with X gal in media
DNA is cloned into plasmid restriction site within lacZ gene but when it's being interrupted by an inserted gene = lacZ cannot produce Beta galactosidase
What are the 3 stages of the PCR cycle?
Denaturation, Annealing, & Extension
Who and when was the polymerase chain reaction (PCR) created?
Developed in 1983 by Kary Mullis (1944-2019)
Which colonies did not receive a plasmid? A. 2, 4, 5 B. 1, 3, 6 C. 1 & 2 D. All of the colonies E. None of the colonies
E. None of the colonies b/c : The plate has ampicillin and only the bacteria that have the plasmid could survive even if the plasmid recirculaized
An example of DNA library
E. coli
What laboratory technique is used to identify which chromosome a gene interest when generating a karyotype? A.Southern Blot B.Western Blot C.Reverse transcription PCR D.Fluorescence in situ hybridization E.None of the above
Flourescence in situ hybridization
What are the two types of DNA libraries ?
Genomic & Complementary DNA (cDNA) Libraries
What and when was the 2nd recombinant human protein marketed
Growth Hormone in 1985
Why can't you use DNA Pol isolated from bacteria that live optimally @ 37° C?
Human DNA polymerase are designed to work best @ @ 37° C but to separate a double DNA strand, you will need to amplify it with a PCR & denature it by boiling it at 95 ° C
What and when was the 1st recombinant human protein marketed
Insulin in 1982
Assume you want to make lots of human insulin using a bacteria expression vector. Why wouldn't using human insulin genomic DNA for this clone project be advantageous?
Introns and Exons, cDNA comes from mRNA that corresponds to the human gene.
What does it mean if there are overlapping peaks in a electropherogram?
It can mean there is possible -contamination, - mis priming (primers annealing in more than one area instead of just one.), - amplify a gene that is part of multigene, -Or a presence of a pseudogene.
Who and when was southern blotting created
It was developed by Edward Southern in 1975
Colony hybridization
Library screening to identify the gene of interest
What prevents restriction enzymes produced by bacteria from digesting its own genome? A. Restriction preventase B. Medichlorians C. Acetylated histones D. Methylated DNA E. None of the above
Methylated DNA
disadvantage of pcr
Need to know something about the DNA sequence that flanks the gene of interest to design primers
Who & when was the 2nd protein structure discovered?
Pauling & Corey 1951 described 2nd regular 2nd structures
Vectors
Pieces of DNA that can accept, carry, & replicate pieces of DNA
What laboratory technique utilizes a probe with a 5' end reporter and a 3' end quencher to study gene expression? A. Northern blot blot B. Real time PCR C. Reverse transcription D. Flourescence in situ hybridization E. None of the above
Real Time PCR
What laboratory technique is used to study mRNA in a sample when the level of detection is below that of a Northern blot A.Southern Blot B.Western Blot C.Reverse transcription PCR D.Fluorescence in situ hybridization E.None of the above
Reverse transcription PCR
Library screening rarely results in the cloning of full-length gene
Since it only shows sequenced small pieces of a gene; but scientists look for overlapping sequences by looking for the start and stop codons to know when the full length of the gene is obtained
origin of replication
Site for DNA replication that lets plasmid to replicate from the chromosome
What additional enzymes would need to be mass produced for recombinant DNA technology?
Taq polymerase, restriction enzymes, DNA ligase. reverse transcriptase nucleases, endonucleases EcoR1, & EcoRV
2 ways to study gene expression are
Tawan Probes & SYBR GREEN
Genomic DNA Library
The collection of chromosomal DNA molecules from the tissue of interest is isolated & digested with a restriction enzyme which produces many fragments that include the entire genome
Assume the human genome project was not complete but you wanted clone growth hormone from humans, what sequence would you, you use to design PCR primers?
The flanking regions associated with mouse with mouse DNA to design the forward and reverse primers. Hopefully the mouse is similar enough, and well have success in amplifying the human hormone
Primary Structure
The sequence amino acids are linked together
T / F: The type of probe used depends on what is already known about the gene of interest
True
T \ F: cloning PCR products is > using DNA library since its faster & more effective
True
T/ F Each cycle consists of three stages
True
Southern Blotting
Used to determine gene copy number; gene mapping; gene mutation detection; PCR product confirmation
Why do we clone PCR products in a electropherogram?
We use it to avoid overlapping peaks
Secondary Structure
When aminco acids twist / fold at a specific points to create shape based on the formation between hydrogen bonds and hydrophobic acids
How does a functional lacZ become a blue colony?
When an X gal is added to media in Petri plate
If gene sequence has NOT been cloned in another species but something is known about the protein, what can be done?
You can work backwards by figuring out what the amino acid sequence of the protein is
DNA Sequencing: Sanger Method
a method of chain-termination sequencing that determines the sequence of nucleotides of a cloned gene that was developed by Frederick Sanger in 1977
Selection
a process that identifies recombinant bacteria from bacteria that has plasmid without foreign DNA while preventing the growth of nontransformed bacteria
DNA Sequence involves
a reaction tube containing: o Single primer annealing to denatured DNA template o All 4 dNTPs o DNA Polymerase o Dideoxynucleotide (ddNTP) which has a 3' H instead of 3' OH on the deoxyribose so it cannot form a phosphodiester bond with the incoming nucleotide & so gets terminated.
Taq polymerase puts
a single adenine nucleotide on the 3' end of all PCR products
Plasmid DNA
a small circular piece of DNA found in bacteria
nothern blot
a technique that involves analyzing mRNA produced by a tissue & used to study gene expression
Calcium Chloride Transformation of Bacterial Cells
an inefficient process that inserts bacteria into DNA cells
What does fluorescence on more than one chromosome indicate?
analyze the possibility of genetic disorders and determine which cells in a particular organ or tissue are expressing the particular mRNA
Electroporation
applying a brief high voltage pulseto create temporary holes in the bacteria's cell wall that allows DNA to enter
DNA Libraries can
be screened to pick out different genes of interest
SYBR green
binds double stranded DNA, and as more double stranded DNA is copied with each round of qPCR there are more DNA copies to bind SYBR Green which increases the amount of fluorescent light emitted
(modern) Sanger method uses
capillary electrophoresis that enables greater than 600 nucleotides to be sequenced per reaction
Tawman probes are
complimentary to specific regions of target cDNA between forward and reverse primers for PCR
Tawman probes
contain reporter located at 5' end of probe and can emit fluorescent light when excited by the laser without the quencher which is attached to 3' end of probe
Denaturation (PCR)
heat to 94 ° C to 96° C
Non-functional lac Z
is a white colony produces cloned bacteria cells with pairs of recombinant plasmid
Taq DNA polymerase
isolated from a species known as Thermus aquaticus that thrive in hot springs
disadvantages of plant upstream processing
not all proteins can be expressed in plants-cell wall-process of glycosylation is slightly different from animal cells
314 Chip
o 1.3 million wells o 30-100Mb Gb o 400-500 thousand reads o $1,100.00
318 Chip
o 11 million wells o 600 Mb- 2.0 Gb o 4-5.5 million reads o $1,500.00
316 Chip
o 6.3 million wells o 300 Mb-1.0 Gb o 2-3 million reads o $1,300.00
What is the procedure of the (modern) Sangar Method
only uses 1 reaction tube instead of 4
what was the source of growth hormone prior to recombinant technology?
pituitary glands of cadavers
Annealing (PCR)
primers H bond with complementary bases at the opposite ends of the target sequence at 52° C to 58° C.
multiple cloning site
recognition site for restricted enzymes which DNA inserted & cloned into
Antibiotic Selection
transformed cells are cultured on plates containing antibiotics to identify recombinant bacteria & nontransformed bacteria
RNA polymerase promoter sequences
used for transcription in vitro and in vivo
Reverse Transcription PCR
used to study mRNA levels when level of detection is below that of Northern
Hydrophobic
water hating
Hydrophilic
water loving
Clone human insulin DNA sequence into plasmid & bacteria cells
were used to synthesize protein of the cloned gene
advantages of PCR
• Amplify millions of copies of target DNA from small amounts of starting material in short period of time • To calculate the number of copies of target DNA starting with 1 molecule of DNA using this equation 2n in which N represents the number of PCB cycles
Proteins have
• Complex molecules built of chains of amino acids • specific molecular weights • electrical charge that causes them to interact with other molecules
NGS (2ndGS) with Post Light Sequencing
• Ion Torrent PGM • Utilizes release of H+ on semiconductor chip • DNA → Ions → Sequence • One sensor per well per sequencing reaction • Direct detection of natural DNA extension • Millions of sequencing reactions per chip.
3rd Generation Sequencing with single molecule reads
• Oxfod Nanopore Technologies • MinIon reads single molecules • 10+ Kb, error rate approximately 5% • Sensor detects changes in ionic current at nanopore
