biotechnology chapter 3- quiz 3 and 4

Plasmid DNA

small circular pieces of DNA found primarily in bacteria. -considered extrachromosomal DNA -Can be used as vectors

Proteomics

studying all proteins in a cell

glycomics

studying carbohydrates of a cell

Metabalomics

studying proteins and enzymatic pathways involved in cell metabolism

reverse transcription

synthesis of DNA from an RNA template -retro virus -reverse transcriptase

DNA Pol

synthesizes second strand of DNA to create double stranded cDNA

Hybridization of DNA

takes ssDNA back to dsDNA. Takes away the denaturant.

PCG annealing

(hybridization) - in which primers H bond with complementary bases at the opposite ends of target sequence at 55 °C to 65 °C

PCR stages

denaturation, annealing, extension

multiple cloning site (MCS)

recognition sites for several restriction enzymes in which insert is cloned into

Transcriptomics

studying all genes transcribed in a cell

RNA polymerase promoter sequence

used for transcription in vitro and in vivo

transformation of bacterial cells

- A very inefficient process - Process for inserting foreign DNA into bacteria • Treat bacterial cells with calcium chloride • Add plasmid DNA to cells chilled on ice • Heat the cell and DNA mixture • Plasmid DNA enters bacterial cells and is replicated and express their genes

Colony hybridization steps

- Bacterial colonies containing recombinant DNA are grown on an agar plate - Nylon or nitrocellulose filter is placed over the plate and some of the bacterial colonies stick to the filter at the exact location they were on the plate - Treat filter with alkaline solution to lyse the cells and denature the DNA - 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 • DNA fragment that is complementary to the gene of interest - Probe binds by hydrogen bonding to complementary sequences on the filter = hybridization - Filter is washed to remove excess unbound probe - Filter is exposed to film - autoradiography • Anywhere probe has bound to the filter, radioactivity from the radioactive probe or released light (fluorescence or chemiluminescence) from non-radioactive probes exposes silver grains in the film - Depending on the abundance of the gene of interest there might be few colonies or plaques on the filter that hybridize to the probe • Film is developed to create a permanent record of the colony hybridization - Use digital instrument to detect probe binding if a fluorescent or chemiluminescent probe was used - Film is then compared to the original agar plate to identify which colonies contained recombinant plasmid with the gene of interest

DNA library

- Collections of cloned DNA fragments from a particular organism contained within bacteria or viruses as the host - Screened to pick out different genes of interest Two types: - Genomic DNA libraries - Complementary DNA libraries (cDNA libraries)

restriction mapping gene structure

- Cut cloned gene with restriction enzymes to pinpoint location of the cutting sites - Knowing restriction map is useful for making clones of small pieces of the DNA which is called subcloning - These small pieces of DNA can then be sequenced - Protocol of restriction mapping: a. Digest DNA with single or double restriction enzymes b. Separate DNA fragments via agarose gel electrophoresis c. Arrange fragments in order to make map of restriction sites

Blue-white selection

- DNA is cloned into the restriction site in the lacZ gene - When it is interrupted by an inserted gene, the lacZ gene cannot produce functional b - gal - When Xgal (artificial lactose) is added to the plate, if functional lacZ is present = blue colony - Non-functional lacZ = white colony = clone comprising genetically identical bacterial cells each containing copies of recombinant plasmid **you want the white cells because they have taken on the foreign DNA **you do not want the blue ones because they have closed in on themselves and have preserved B-Gal and Lac Z which means they did not take on the foreign DNA

Original Sanger method

- Had four separate reaction tubes and each contained the vector; primer; dNTPs in which one dNTP is radioactively labeled; and a different small amount of ddNTP; DNA Pol - Over time a ddNTP will be incorporated into all the positions in the newly synthesized strands creating fragments of varying lengths that are terminated at the ddNTP - Then the fragments are separated on polyacryamide gel - Autoradiography used to then identify radioactive fragments - Read the gel from bottom to top as individual nucleotides - The sequence generated from the reaction is complimentary to the sequence on the template strand in the vector

DNA sequencing

- Important to determine the sequence of nucleotides of the cloned gene - Why it is important to know the sequence of the cloned DNA? - Chain termination sequencing (Sanger method) - Requires single stranded primer annealing to denatured DNA template and then the reaction tube also contains all 4 dNTPs; DNA Pol, and 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 and so gets terminated

comparative genomics

- Mapping and sequencing genomes from a number of model organisms - Allows researchers to study gene structure and function in these organisms in ways designed to understand gene structure and function in other species including humans

How to clone a gene of interest

- mRNA from tissue of interest is isolated - Need to make double stranded DNA from mRNA: How? a. enzyme reverse transcriptase catalyzes synthesis of complementary single stranded DNA from mRNA i. Called complementary DNA (cDNA) because it is an exact copy of the mRNA b. mRNA is degraded either with an enzyme or alklaline solution c. DNA Pol is used to synthesize second strand of DNA to create double stranded cDNA - Short linker double stranded DNA sequences which contain restriction enzyme recognition sites are added to the ends of the cDNA - Cut with restriction enzyme, cut vector with same enzyme, ligate fragments to create recombinant vectors - Then transform bacteria with recombinant vectors

factors in deciding the temp needed to denature DNA

-number of G-C pairs -what denaturant is being used -length of strand -mismatch of base pairs

origin of replication (ori)

-site for DNA replication that allow plasmids to replicate independently from host chromosome • Copy number: number of plasmids in the cell (normally small but plasmids have high copy numbers)

Features of a good vector

-size -origin of replication -multiple cloning site -selectable marker genes -RNA polymerase promoter sequence -DNA sequencing primers

Recognition site

4 or 6 bp cutters are so named because they recognize restriction sites with a sequence of 4 or 6 nucleotides

cDNA library

A gene library containing clones that carry complementary DNA (cDNA) inserts. The library includes only the genes that were transcribed in the cells whose mRNA was isolated to make the cDNA.

RISC (RNA-induced silencing complex)

A protein complex that is targeted to specific mRNA molecules by base pairing with short regions on the target mRNA, inhibiting translation or degrading the RNA.

restriction site

A specific sequence on a DNA strand that is recognized as a cut site by a restriction enzyme. - is a palindrome - reads same forward and backward on opposite strands of DNA

electroporation

A technique to introduce recombinant DNA into cells by applying a brief electrical pulse to a solution containing the cells. The pulse creates temporary holes in the cells' plasma membrane, through which DNA can enter.

palindrome

A word or an expression that is spelled the same backward and forward **MIRROR IMAGE**

reverse transcriptase

An enzyme encoded by some certain viruses (retroviruses) that uses RNA as a template for DNA synthesis. -turns RNA back into DNA

restriction enzymes

DNA cutting enzymes (molecular scissors). -Primarily found in bacteria - Cut DNA by cleaving the phosphodiester bond that joins adjacent nucleotides in a DNA strand - Bind to, recognize, and cut DNA within specific sequences of bases called a restriction site

third generation sequencing

DNA polymerase adds fluorescently tagged nucleotides to synthesize DNA. Fluorescent tag is cleaved off each base as it is added to the DNA strand. -Each base added emits a characteristic fluorescent color that can be detected.

Recombinant DNA

DNA produced by combining DNA from different sources

Bioinformatics

Mergingmolecularbiologywith computer technology - An interdisciplinary field that applies computer science and information technology to promote an understanding of biological processes

cohesive (sticky) ends

Overhanging single-stranded ends of a DNA molecule created by the action of certain restriction enzymes. - Preferred for cloning because DNA fragments with sticky ends can be easily joined together because they base pair with each other by forming weak hydrogen bonds

Real-time PCR (qPCR)

PCR based method which is used to amplify and quantify a targeted DNA molecule. -Can quantify amplification reactions as they occur in real time by use of Taqman probes. This allows you to ensure that your duplicates are being created and amplified correctly.

PCR (polymerase chain reaction)

Technique for making copies, or amplifying, a specific sequence of DNA in a short period of time. -genomic DNA comes apart at 95 degrees celsius -our DNA is only stable at ~ 37 degrees celsius Taq Pol is used to thermostabilize our DNA -each cycle consists of 3 stages **basically you heat DNA to separate it, then cool it to bring it back together, and then heat it again. -Using a gene from the library, you can build many many genes in a small amount of time. **The type of DNA polymerase used is very important - Taq DNA polymerase - isolated from a species known as Thermus aquaticus that thrives in hot springs - Cannot use DNA Pol isolated from bacteria that live at 37 C because it will denature at DNA melting temp

Northern blotting

Used for RNA sequencing • Basic method is similar to Southern blotting • RNA is isolated from a tissue of interest, separated by gel electrophoresis, blotted onto a membrane, and hybridized to a labeled DNA probe, exposed bands on autoradiograph show presence of mRNA for gene of interest as well as size of mRNA • Can compare and quantify amounts of mRNA present in different tissues

Southern blotting

Used fro DNA sequencing • Digest chromosomal DNA into small fragments with restriction enzymes • Fragments are separated by agarose gel electrophoresis • Gel is treated with alkaline solution to denature the DNA • Fragments are transferred onto a nylon or nitrocellulose filter (called blotting) • Filter (blot) is baked or exposed to UV light to permanently attach the DNA • Filter (blot) is incubated with a labeled probe and exposed to film by autoradiography • Number of bands on film represents gene copy number -used to determine gene copy number; gene mapping; gene mutation detection; PCR product confirmation; DNA fingerprinting

Selection

a process designed to facilitate the identification of recombinant bacteria while preventing the growth of non-transformed bacteria and bacteria that contain plasmid without foreign DNA

Smal

a restriction enzyme that cuts DNA with the sequence CCCGGG and creates blunt ends. -cuts between between C and G

EcoRI

a restriction enzyme that specifically cuts DNA with sequence GAATTC and creates sticky ends. -Cuts between G and A on the template strand -Cuts between A dnd G on the complimentary strand

selectable marker gene

allow to select for transformed colonies -must have a way to find the needle in the haystack

Vector

allow you to take DNA and move it inside of a bacteria. *suitcase* -plasmids are number one used form

restriction enzyme cuts

a. Some cut DNA to create DNA fragments with overhanging single stranded ends called "sticky" or "cohesive" ends b. Some cut DNA to generate fragments with double-stranded ends called "blunt" ends

metagenomics

analysis of genomes of organisms in an environment

Plasmid DNA Vectors

circular form of self-replicating DNA • Can be manipulated to carry and clone other pieces of DNA

TaqMan probes in RT-PCR

complementarytospecific regions of target DNA between forward and reverse primers for PCR - Taqman probes contain two dyes: reporter located at 5' end of probe and can release fluorescent light when excited by the laser and other dye is quencher which is attached to 3' end of probe

pharmacogenomics

customized medicine based on person's genetic profile for a particular condition

interactome

describes the interacting components of a cell

network map

diagram of interacting proteins, genes and other molecules

short linker

double stranded DNA sequences which contain restriction enzyme recognition sites are added to the ends of the cDNA

PCR Extension

increase temperature - add DNA polymerase and nucleotides to produce two complete strands -elongation) - DNA Pol copies target DNA at 70 to 75 °C

first human protein expressed via recombinant technique

insulin followed by HGH

Nutrigenomics

interaction between genes and diet

systems biology

interprets genomic information in the context of the structure function and regulation of biological pathways. Can be applied to cells or to an entire organism

disadvantage of blunt end cutter like Smal1

is that there will be no overlap and no hydrogen bonding ends float apart

poly linker

multiple cloning site

RNA interference (RNAi)

naturally occurring mechanism for inhibiting gene expression. - Double stranded RNA can be bound by Dicer enzyme that cuts dsRNA into 21-25 nucleotide snippets called small interfering RNA (siRNA) - siRNA then bound to protein: RNA complex called RNA Inducins silencing complex (RISC) - RISC unwinds dsRNA releasing single stranded RNA that bind complementary mRNA - Binding of siRNA to mRNA leads to degradation of mRNA by Slicer enzyme or blocks translation by interfering with ribosome binding - Currently developing techniques to use RNAi to silence gene expression

Vector size

needs to be small enough to be separated from chromosomal DNA of host plasmid

Bacteriophage vectors

only simple types of proteins can be made in bacteria because of their lack of organelles. They don't process things the way we do. -bacteria aren't big enough to handle the size of our genes.

Antibiotic Selection

plate transformed cells on plates containing different antibiotics to identify recombinant bacteria and non-transformed bacteria - Does not select for plasmid containing foreign DNA vs. recircularized plasmid

GenBank

public database of DNA sequences and contains National Institute of Health collection of DNA sequences

agarose gel electrophoresis

separate and visualize DNA fragments based on size • Agarose is isolated from seaweed and when melted in a buffer solution and poured into a horizontal tray and as it cools it will form a semisolid gel containing small pores through which DNA will travel -- To run a gel, it is submerged in a buffer solution that conducts electricity - DNA is loaded into small depressions called wells at the top of the gel - Electric current is applied through electrodes at opposite ends of the gel • DNA migrates according to its charge and size • Rate of migration through the gel depends on the size of the DNA because the sugar phosphate backbone makes it always negatively charged • DNA migrates toward positive pole and is repelled by negative pole • Migrationdistanceisinverselyproportionaltosize of DNA fragment - Large fragments migrate slowly; smaller fragments migrate faster - Tracking dye is added to the samples to monitor DNA migration during electrophoresis - DNA can be visualized after electrophoresis by the addition of DNA staining dyes • Ethidium bromide: intercalate between DNA base pairs and it fluoresces under ultraviolet light • Then a picture can be taken to document the gel results

PCR Denaturation

the DNA is heated to break the hydrogen bonds and pull the strands apart. -heat to 94 °C to 96 °C

reverse transcription PCR

used to study mRNA levels when level detection is below that of the Northern procedure. Procedure: • 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 fragments • Determine expression patterns in the tissue • Amount of cDNA produced in RT PCR reaction for gene of interest reflects amount of mRNA and level of gene expression

viral plaque

visible area where the virus has destroyed infected cells in a cell culture

Advantage of cDNA library

• Collection of actively expressed genes in the cells or tissues from which the mRNA was isolated • Introns are NOT cloned • Can be created and screened to isolate genes that are primarily expressed only under certain conditions in a tissue

disadvantages of genomic libraries

• Introns are cloned in addition to exons; - Majority of genomic DNA is introns in eukaryotes so majority of the library will contain non-coding pieces of DNA • Many organisms have very large genome, so searching for gene of interest is difficult • Time consuming!