3. Recombinant DNA Technology and Genomics

Advantage and disadvantage of Cloning PCR products

Adv: rapid and effective compared to using DNA libraries. Disadv: need to know something about the DNA sequence that flanks the gene of interest to design primers

Restriction enzymes don't digest bacteria DNA

Bacteria DNA are methylated. Restriction enzymes cannot cleave methylated DNA

Types of Vectors

Bacterial plasmid vectors Bacteriophage vectors Cosmid vectors Expression vectors Bacterial Artificial Chromosomes (BAC) Yeast Artificial Chromosomes (YAC) Ti vectors

Features of DNA cloning vectors

Small size Origin of replication (ori) Multiple cloning site (MCS) Selectable marker genes RNA polymerase promoter sequences DNA sequencing primers

Protection from bacteriophages

Some bacteria have enzymes that can cut viral DNA into pieces, thus preventing viral replication (restriction enzymes)

Origin of replication (ori)

site for DNA replication that allow plasmids to replicate independently from host chromosome.

What we have learned from HGP

* Many human genes are capable of making more than one protein, allowing human cells to make at least 100,000 proteins from only about 20,000 genes. • Chromosome 1 contains the highest number of genes. The Y chromosome contains the fewest genes. • Many of the genes in the human genome show a high degree of sequence similarity to genes in other organisms. • Thousands of human disease genes have been identified and mapped to their chromosomal locations

Projects after HGP

- Human epigenome project—create maps of epigenetic changes in different cell and tissue types and evaluate potential roles of epigenetics in complex diseases - International Hapmap Project—characterization of SNPs for their role in genome variation, disease and pharacogenomics applications - Encyclopedia of DNA elements (ENCODE)—use experimental approaches and bioinformatics to ID and analyze functional elements that regulate expression of human genes - Personalized genome projects - Cancer genome project—map important genes and genetic changes involved in cancer. identification of key genes involved in tumor formation and metastasis (spreading) will lead to imporved diagnostic techniques for the detection of cancer and more effective treatments for curing cancer.

The Human Genome Project (HGP)

- Started in 1990 by the U.S. Department of Energy - International collaborative effort to identify all human genes and to sequence all the base pairs of the 24 human chromosomes - Mostly done by 20 centers in 18 countries: China, France, Germany, Great Britain, Japan, and the United States - Competitor was a private company, Celera Genomics, directed by Dr. J. Craig Venter - April 14, 2003: map of the human genome was completed - Consists of 20,000 protein-coding genes - Map was complete with virtually all bases identified and placed in their order and potential genes assigned to chromosomes

Applications of recombinant DNA technology

- use purified protein to make antibodies for medical purpose and/or make vaccines for the treatment of disease - find chromosomal location of cloned gene, determine gene copy number, and study gene structure - mutate gene and study function of altered protein produced - create new, genetically engineered microorganisms, animals, and plants with a range of applications from waste-degrading microorganisms to disease-resistant plants and animals - use in forensic applications such as DNA fingerprinting - diagnose human genetic disorders and infectious disease conditions. - use in human gene therapy - scale-up production, isolation, and purification of therapeutic proteins for use in humans as recombinant DNA products - create transgenic animals and gene knock-out animals to study gene function - study gene structure, gene sequence, and gene expression in organs, tissues, and individual cells - express protein and study protein structure and function.

RNA interference (RNAi) silence expression of genes

1) Dicer enzyme cuts dsRNA into 21-25 nucleotide snippets [small interfering RNA (siRNA)] 2) RNA inducins silencing complex (RISC) protein bind siRNAs and degrade one of the two strands. 3) RISC unwinds dsRNA releasing single stranded siRNA that binds to complementary mRNA in the cytoplasm 4) Binding of single stranded siRNA to mRNA leads to degradation of mRNA by Slicer enzyme or blocks translation of mRNA by interfering with ribosome binding. Currently developing techniques to use RNAi to silence gene expression

Protocol of restriction mapping

1) Digest DNA with single or double restriction enzymes (single digests with restriction enzymes, double digests with combined enzymes) 2) separate DNA fragments via agarose gel electrophoresis. 3) Arrange fragments in order to make map of restriction sites. * Scientists today use bioinformatic software to identify restriction sites in the DNA

Southern blotting procedure

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 • Number of bands on film represents gene copy number

Important to know the sequence of the cloned DNA

1) to deduce the amino acid sequence of a protein encoded by a cloned gene 2) to determine the exact structure of gene 3) to identify regulatory elements such as promoter sequences 4) to identify differences in genes created by gene splicing 5) to identify genetic mutations among other reasons.

PCR process

1. Denaturation: heat to 94-96ºC - causing separation of the target DNA into single strands. 2. Annealing (hybridization): primers H bond with complementary bases at the opposite ends of target sequence at 55-65ºC 3. Extension (elongation): DNA polymerase copies target DNA at 70-75ºC by binding to the 3' end of each primer and using the primers as templates. DNA polymerase adds nucleotides to the 3' end of each primer to synthesize a complementary strand. At the end of one cycle, the amount of DNA has doubled. Cycles usually repeated for 20-30 times.

Sanger method

4 separated reaction tube containing vector, primer, dNTPs (one dNTP radioactively labeleD), different small amount of ddNTP, DNA polymerase - ddNTP incorporate into all positions in the newly synthesized strands creating fragments of varying length terminated at the ddNTP - separate fragments on polyacryamide gel - use autoradiography to identify radioactive fragments - read the gel from bottom to top - sequence generated complimentary to the sequence on the template strand in the vector.

High throughput computer automated sequencing

>500 nucleotides to be sequenced per reaction - 1 reaction tube - ddNTPs labeled with different fluorescent dye - separating sequencing reaction - samples separated on a single-lane capillary gel (an ultrathin diameter tube gel) - DNA fragment move through gel - scanned with a laser beam - laser stimulates fluorescent dye on each DNA fragment (different color) - emitted light collected by a detector - amplifies and feeds into computer - computer can run multiple capillary gels at one time (900bp) - computer converts light patterns to reveal sequence. - generate many sequences at a relatively shorter time. Helpful for completing the HGP

Colony hybridization: if gene sequence has not been cloned in another species but something is known about the protein

A series of chemically synthesized oligonucleotides can be made based on a prediction of codons that can code for the known protein sequences. If some partial amino acid sequence is known for a protein encoded by a gene to be cloned, it is possible to work backwards and design oligonucleotides based on the predicted nucleotides that coded for the amino acid sequence. If an antibody is available for the protein encoded by the gene of interest, an expression library, which results in protein expression in bacteria, can be used, and the library can be screened with the antibody to detect colonies expressing the recombinant protein.

Bacteriophage vectors

Allow cloning of larger DNA fragments (up to approx. 25kb). The λ chromosome is a linear structure approx. 49kb in size. Cloned DNA is inserted into restriction sites in center of λ chromosome. Recombinant chromosomes are packaged into viral particles in vitro. These phages then infect lawn of E. coli cells. When they infect E. coli as a host, the λ chromosome uses COS sites (12 base pairs sites) to circularize and then replicate. Bacteriophage λ1 replicates through the lytic cycle. As λ replicates to create more viral particles, infected E. coli are lysed (split/ ruptured) by λ, creating zones of dead bacteria called plaques, which appear as cleared spots on the bacterial lawn. Each plaque contains millions of recombinant phages particles. Application: cDNA, genomic and expression libraries. Limitations: packaging limits DNA insert size; host replication problems.

selectable marker genes

Allow for the selection and identification of bacteria that have been transformed with a recombinant plasmid. e.g. genes for ampicillin resistance (ampR), tetracycline resistance (tetR), lacZ gene for blue-white selection.

Bacteria expression vector

Allow high level protein expression in bacterial cells because they have a prokaryotic promoter sequence enxt to the site where DNA is inserted into the plasmid (MCS). Bacterial RNA polymerase can then bind to promoter and transcribe the insert's sequence which is then translated into protein. Protein is then purified using biochemical techniques. Problem: Bacterial ribosomes sometimes cannot translate eukaryotic mRNA sequences. If a protein is produced, it may not fold and be processed correctly, as bacteria does not have organelles for processing. Also, making some recombinant products in bacteria can be a problem because E. coli often does not secrete proteins; therefore expression vectors are often used in a strain more suitable for protein secretion. In some cases, the host bacteria can recognise recombinant proteins as foreign and degrade the protein, whereas in others the expressed protein is lethal to the host bacterial cells.

Autoradiogram

An autoradiogram from an original dideoxy sequencing reaction in which radioactive ddNTPs or primers were used and four separate reactions carried out. The letters over the lanes (a, C, G and T) correspond to the particular ddNTP used in the sequencing reaction analyzed in the lane. Analysing Sanger sequencing by autoradiography is largely obsolete, having been replaced by computer-automated sequencing approaches and next-generation sequencing technologies

Stone Age Genomics (paleogenomics)

Analyse "ancient" DNA - demonstrates how stable DNA can be under the right conditions, particularly when frozen

Bacterial artificial chromosome (BAC, circular)

Applications: Genomic libraries, cloning large DNA fragments. Used during the HGP to clone and sequence large pieces of chromosomes. Large low copy plasmids. Contain genes that encode the F factor (unit of genes controlling bacterial replication). Can accept large sizes of DNA inserts ranging from 100-300kb. Limitations: replication restricted to bacteria; cannot be used for protein expression.

PCR cloning

As DNA is copied, Taq polymerase puts a single adenine nucleotide on the 3' end of all PCR products. After amplifying the target gene, cloned PCR products can be ligated into plasmids called T vectors. T vector has single stranded thymine on each end can complementary base pair with the adenine in the PCR products. Once ligated into a T vector, the recombinant plasmid containing the cloned PCR product can be introduced into bacteria and its nucleotide sequence can be determined.

Colony hybridization

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 by baking or exposing to UV light. Filter is incubated with a probe that is tagged with a radioactive nucleotide or fluorescent dye. DNA fragment 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 developed to create a permanent record of the colony hybridization (autoradiograph/ autoradiogram). Filter is exposed to X-ray film (autoradiography) - anywhere probe has bound to the filter, radioactivity from the radioactive probe or released light from non-radioactive probes exposes silver grains in the film. Often when hybridization is done using fluorescent or chemiluminescent probes, a digital imaging instrument can be used to detect probe binding and then a photograph aligned with the bacterial plate to identify colonies of interest.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 then compared to the original agar plate to identify which colonies contained recombinant plasmid with the gene of interest.These colonies can now be grown on a large scale to isolate the cloned DNA.

DNA database searching

Basic Local Alignment Search Tool (BLAST) - Used to search GenBank for sequence matches between cloned genes and to create DNA sequence alignments

Advantage of PCR

Can amplify millions of copies of target DNA from small amount of starting material in short period of time.

ABI SOLID (supported oligonucleotide ligation and detection) method

Can produce 6 gigabases of sequence data per run - Similar to 454 approach in which DNA fragments are linked to beads and amplified but different sequencing technologies used - can provide greater output of sequencing data per instrument run

Real time/ 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 dye containing probe or DNA binding dye that emits fluorescent light when illuminated by the laser. Light emitted by the dyes correlates with amount of PCR product amplified. Light is captured 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

Genomic libraries

Chromosomal DNA from the tissue of interest is isolated and digested with a restriction enzyme which produces many fragments that include the entire genome. Vector is digested with the same enzyme. DNA ligase is used to ligate genomic DNA fragments and vector DNA. (in theory, all DNA fragments in the genome will be cloned into a vector). Recombinant vectors are used to transform bacteria and theoretically each bacteria will contain a recombinant plasmid.

Restriction mapping gene structure

Cut cloned gene with restriction enzymes - sequence these DNA protocol of restriction mapping: digest DNA with single/ double restriction enzymes - separate DNA fragments via electrophoresis - arrange fragments to make map of restriction sites.

Applications of PCR

DNA cloning Study gene expression Diagnostic tests for disease-causing pathogens (test samples) Human remains identification Paternity testing and determining family relationships Forensic DNA analysis Amplification of rare DNA (e.g. from fossils) Human genetic testing and disease diagnosis.

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 Beta gal. When Xgal (artificial lactose) is added to the plate, if functional lacZ is present = blue colony. Non-functional lacZ = white colony = clone = genetically identical bacterial cells each containing copies of recombinant plasmid.

Application of Bioinformatics

Databases to store, share, and obtain the maximum amount of information related to gene structure, gene sequence and expression, and protein structure and function. These databases can also be used to predict the sequence of amino acids encoded by a nucleotide sequence and to provide information on the function of the cloned gene.

Polymerase Chain Reaction (PCR)

Developed in the mid- 1980s by Karl Mullis. Technique for making copies, or amplifying, a specific sequence of DNA in a short period of time. Target DNA to be amplified is added to a tube, mixed with nucleotides (dATP, dCTP, dGTP, dTTP), buffer, and DNA polymerase. Paired set of Forward and Reverse Primers are added (short single-stranded DNA oligonucleotides 20-30bp long). Primers are complementary to nucleotides flanking opposite ends of target DNA. Reaction tube is placed in a thermocycler, a sophisticated heating block that is capable of rapidly changing temperature over very short time intervals. The thermocycler will take DNA through the PCR cycle.

Procedure to do array

Each microarray containing unique sequences of DNA for a different gene 1) Extract mRNA from a tissue of interest and cDNA is synthesized from mRNA and labeled with fluorescent dye 2) Labeled cDNA is incubated overnight with the array where it hybridizes with different spot on the array that contain complementary DNA sequences 3) Can have over 10,000 spots of DNA 4) Array is washed and scanned by a laser that causes cDNA hybridized to array to fluoresce 5) Fluorescent spots reveal which genes were regulated/ expressed and Intensity of fluorescence indicates relative amount of gene expression. Protein expression and purification Bacteria that have been transformed with recombinant plasmids can often be used to produce the protein product of the isolated gene. large quantities of bacteria can be grown in a fermenter, and the protein can be isolated.

Gene microarray (gene chip)

Enable researchers to study all of the genes expressed in a tissue very fast. created with use of small glass microscope slide. Single stranded DNA molecules are spotted on the slide using an arrayer (computer controlled robotic arm) which fixes DNA (multiple copies of cDNA) at different spots on the slide which is recorded by a computer. an arrayer is fitted with a number of tiny pins. Each pin is immersed in a small amount of solution containing millions of copies of different DNA molecules, such as cDNAs for different genes.

Calculate number of copies of target DNA starting with 1 molecule of DNA

Equation: 2^n (n = number of PCR cycles)

Genomics and bioinformaticts

Genomic DNA cut into smaller pieces (labeled 1,2,3,4) by EcoRI and BamHI. Regardless of how a piece of DNA is cloned, DNA sequence analysis is an essential part of learning more about the cloned DNA a) typically such analysis begins by searching the unknown sequence against a database of known sequences. For simplicity, only one strand of sequenced DNA is being compared with the database b) Another application of bioinformatics involves the use of computer programs to align DNA fragments (contigs) based on nucleotide sequence overlaps. When contigs are compared, typically only relatively short sequences at the end of each contig are aligned to assemble an entire chromosome.

Types of DNA libraries

Genomic DNA libraries Complementary DNA (cDNA) libraries

DNA sequencing

Important to determine the sequence of nucleotides of the cloned gene.

FISH: fluorescence on more than one chromosome

Indicates either multiple copies of the gene or related sequences that may be part of a gene family.

Transformation of bacterial cells (?)

Inefficient 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.

First human protein expressed via recombinant techniques

Insulin: clone human insulin DNA sequence into a plasmid and the bacterial cells were then used to synthesize the protein product of the cloned gene. growth hormone

Disadvantages of genomic libraries

Introns are cloned in addition to exons - majority of genomic DNA is introns so majority of the library will contain non-coding pieces of DNA.] Many organisms have very large genome, so search for gene of interest is difficult and time consuming. (bacterial cells cannot splice mRNA and remove introns)

Yeast artificial chromosome (YAC, circular)

Is miniature version of eukaryotic chromosome containing origin of replication, two telomeres, selectable markers, centromere; that allows replication of YAC and segregation into daughter cells during cell division. Best for cloning very large DNA inserts from 200kb to 2 megabases. Applications: genomic libraries, cloning large DNA fragments, used for HGP Limitations: must be grown in yeast; cannot be used in bacteria

Source of growth hormone prior to recombinant technology

Isolated from tissues. E.g. growth hormone isolated from the pituitary gland of human cadavers. This process was expensive and inefficient, and may have a risk of unknowingly co-purifying viruses and other pathogens as contaminants that could be passed to people receiving the hormone.

Migration distance is inversely proportional to size 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 penetrate between DNA base pairs and it fluoresces under UV light. A picture can be taken to document the gel results.

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

Bioinformatics

Merging molecular biology with computer technology - An interdisciplinary field that applies computer science and information technology to promote an understanding of biological processes

Ti vector (circular)

Naturally occuring plasmids isolated from the bacterium that is a soil plant pathogen causing disease in plants. When the bacteria infects plant cells, the T DNA from the Ti plasmid inserts into the host chromosome. T DNA codes for auxin hormones that weakens plant cell wall and infected plants divided and enlarge to form a tumor (gall). Applications: remove auxin and other detrimental genes from the Ti plasmid, so that the resulting vector could be used to deliver genes into plant cells. Limitations: limited to plant cells only; number of restriction sites randomly distributed; large size of vector not easily manipulated

Findings from HGP

Nearly 50% genes do not yet have a function. The human genome consists of approximately 3.1 billion base pairs - The genome is approximately 99.9% the same between individuals of all nationalities - Single-nucleotide polymorphisms (SNPs) and copy number variations (CNVs)—such as long deletions, insertions and duplications in the genome—account for much of the genome diversity identified between humans - Less than 2% of the genome codes for genes - The vast majority of our DNA is non-protein coding, and repetitive DNA sequences account for at least 50% of the noncoding DNA - The genome contains approximately 20,000 protein-coding genes

Advantage of sticky ends

Preferred for cloning because DNA fragments witch sticky ends can be easily joined together - base pair with each other by forming weak hydrogen bonds.

Restriction enzymes

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 (restriction site). Typically 4 or 6 base pair cutters because they recognize restriction sites with a sequence of 4 or 6 nucleotides.

Colony hybridization: tag the probe with either a radioactive nucleotide or fluorescent dye to catalyze light releasing reactions

Probes are tagged or labeled using either a radioactive nucleotide, or more commonly a fluorescent dye or other compounds that can be used to catalyse light - releasing reactions called chemiluminescence. The dye make it possible to track the probe to determine where it binds.

The "omics"

Proteomics—studying all proteins in a cell • Metabolomics—studying proteins and enzymatic pathways involved in cell metabolism • Glycomics—studying carbohydrates of a cell • Transcriptomics—studying all genes transcribed in a cell • Metagenomics—analysis of genomes of organisms in an environment • Pharmacogenomics—customized medicine based on person's genetic profile for a particular condition • Nutrigenomics—interaction between genes and diet * Metabonomics: measuring metabolic products produced by cells in response to stimuli (such as drug treatment) and genetic manipulation

Sticky (Cohesive) ends

Restriction enzymes cut DNA to create DNA fragments with overhanging single stranded ends

Blunt ends

Restriction enzymes cut DNA to generate fragments with double-stranded ends.

Two types of NGS approaches

Roche 454 commercial system using pyrosequencing. ABI SOLID (supported oligonucleotide ligation and detection) method

Northern blot analysis

Study gene expression by analyzing mRNA produced by a tissue. 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

Type of DNA polymerase used in PCR

Taq DNA polymerase: isolated from Thermus aquaticus. T. aquaticus is adapted to live in hot water, it has evolved a DNA polymerase that can withstand high temperature, thus it can withstand the temperature changes necessary for PCR without being denatured.

Studying Gene expression using qPCR: 2 approaches

Taqman probes SYBR green

Expressing a protein via recombinant techniques

The cloned DNA fragment is a gene that encodes a protein product, bacterial cells could be used to synthesize the protein product of the cloned gene. Can generate lots of pure protein via this technique

Agarose Gel electrophoresis

The gel is submerged in a bugger solution that conducts electricity. DNA is loaded into small depressions (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. The sugar phosphate backbone makes DNA negatively charged. DNA migrates toward positive pole (anode) and is repelled by the negative pole (cathode).

Only 20,000 genes code for protein, but it was predicted that there would be 100,000 genes

The prediction was based primarily on estimates that human cells make approx. 100,000 to 150,000 proteins. One reason why the actual number of genes is so much lower than the predicted number is the discovery of large numbers of gene families with related functions. In addition, many genes that code for multiple proteins through alternative splicing have been found. It has been estimated that 92% to 95% of human genes produce multiple proteins through alternative splicing.

Gene mutagenesis studies

To study structure and function of protein produced by a specific gene 1) Site directed mutagenesis: mutations created in specific nucleotides of a cloned gene contained in a vector 2) Gene is then expressed in cells which results in translation of a mutated protein This procedure lets researchers study effects of mutation on protein structure as well as provides information about which nucleotides are important for specific functions of a protein. Very useful in helping identify critical sequences in a gene that produced proteins involved in disease

Whole genome shotgun sequencing or shotgun cloning

Use restriction enzymes to digest pieces of entire chromosomes: - Produces thousands of overlapping fragments called contigs which are each sequenced. - Computer programs are used to align the sequenced fragments based on overlapping sequence pieces Enables reconstruction of the entire sequence of a whole chormosome

Fluorescence in situ hybridization (FISH)

Used to anaylze genetic disorders. Used to determine which cells in a particular organ are expressing the particular mRNA. chromosomes. isolated and spread out on microscope slide - DNA/ RNA probe for gene of interest labeled with fluorescent nucleotides and incubated in solution with slide - slide washed and exposed to fluorescent light- probe bounded to chromosom illuminates - karyotype to determine which chromosome fluorescence Application: can determine if a developing fetus has an abnormal number of chromosomes.

Southern blotting

Used to determine gene copy number; gene mapping; gene mutation detection; PCR product confirmation; DNA fingerprinting

Reverse transcription PCR

Used to study mRNA levels when level of detection is below that of Northern. 1) Isolate mRNA and use Reverse Transcriptase to make double stranded cDNA 2) Use PCR to amplify region of cDNA with set of primers specific for gene of interest 3) Run agarose gel to separate amplified fragments 4) Determine expression patterns in the tissue **Amount of cDNA produced in RT PCR rxn for gene of interest reflects amount of mRNA and level of gene expression

Clone

a molecule, cell, or organism that was produced from another single entity.

Restriction site

a palindrome - reads same forward and backwards on opposite strands of DNA (e.g. madam forward and backward is the same)

Multiple cloning site (MCS)

a segment of DNA with recognition sites for different common restriction enzymes. An MCS provides for great flexibility in the range of DNA fragments that can be cloned into a plasmid because it is possible to insert DNA fragments generated by cutting with many different enzymes

DNA sequencing primers

allow nucleotide sequencing of cloned DNA fragment that have been inserted into the plasmid

high % gel (2%)

allows to resolve smaller size fragments. easier for them to move through the pores than large fragments, which do not separate through the dense gel very well

Electroporation

apply brief pulse of high voltage electricity to create tiny holes in the bacterial cell wall that allow the DNA to enter.

Roche 454 commercial system using pyrosequencing

beads attached to fragmented DNA which is PCR amplified in separate water droplets in oil - beads loaded into multiwell plates and mixed with DNA polymerase - single dNTP flowed over the wells - a pyrophosphate is released when dNTP is incorporated into complementary sequencing strand (make phosphodiester bond) - chemiluminescent reaction take place - produce light with firefly luciferase enzyme - emitted light captured and recorded to determine when a single nucleotide is added to the growing strand. - repeat the nucleotide flow step with each of the 4dNTPs to optain lots of reads of approx. 400 nucleotides and order of 400 million bases of data per 10 hour run.

SYBR green

binds double stranded DNA. as more double stranded DNA is copied with each round of qPCR, there are more DNA copies to bind SYBR Green, which increases amount of fluorescent light emitted

Disadvantages of cDNA libraries

can be difficult to make the cDNA library if a source tissue with an abundant amount of mRNA for the gene is not available

Plasmid DNA

circular form of self-replicating DNA. extrachromosomal DNA: in the cytoplasm in addition to the bacteria chromosome. small: approx 1 - 4 kb Can act as vectors

Genomics

cloning, sequencing, and analyzing entire genomes

cDNA libraries advantage

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.

Creating DNA libraries

collections of cloned DNA fragments from a particular organism contained within bacteria or viruses as the host. Screened to pic out different genes of interest

Taqman probes

complimentary to specific 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 1) The TaqMan method of real-time PCR involves a pair of PCR primers along with a probe sequence complementary to the target gene. The probe contain a reporter dye (R) at one end and a quencher dye (Q) at the other. When the quencher dye is close to the reporter dye, it interferes with fluorescence released by the reporter dye. When Taw DNA polymerase extends a primer to synthesize a strand of DNA, it cleaves the reporter dye off the probe, allowing the reporter to give off energy. 2) Each subsequent PCR cycle removes more reporter dyes, so that increased light emitted from the dye can be captured by a computer to produce a read out of fluorescence intensity with each cycle.

Cosmid vectors

contain COS ends of λ DNA Plasmid origin of replication gene for antibiotic resistance DNA is cloned into restriction site, then cosmid is packaged into viral particles and used to infect E. coli cells at low copy number (most of the viral genes have been removed). Bacterial colonies are grown on the plate and recombinants are screened by antibiotic selection. Advantages: clone fragments between 20-40kb. Application: cDNA and genomic libraries, cloning large DNA fragments. Limitation: phage packaging restrictions; not ideal for protein expression; cannot be replicated in mammalian cells.

Next generation sequencing (NGS)

designed to produce highly accurate and long stretches of DNA sequence (greater than 1 gigabase) per reaction. Is done at very low cost Use parallel formats that use fluorescence imaging techniques

Selection of recombinant bacteria after transformation

facilitate the identification of recombinant bacteria while preventing the growth of non-transforemd bacteria and bacteria that contain plasmid without foreign DNA.

Recombinant DNA Advisory Committee (RAC)

formed in 1975 by NIH (National Institutes of Health). Purpose: evaluate recombinant technology and establish guidlines for research.

cDNA libraries

mRNA from tissue of interest is isolated and made into double stranded DNA by reverse transcription (mRNA cannot be directly cut with restriction enzymes). Reverse transcriptase (enzyme made by retroviruses which converts RNA to DNA) catalyses synthesis of complementary single stranded DNA (cDNA) from mRNA (exact copy of the mRNA). mRNA is degraded either with an enzyme or alkaline solution. DNA polymerase 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. cDNA and vector cut with restriction enzyme, and ligate the fragments to create recombinant vectors

gel % range from 0.5-2%

most application generally

RNA interference (RNAi)

naturally occurring mechanism for inhibiting gene expression

copy number

number of plasmids in the cell

Vectors

pieces of DNA that can accept, carry, and replicate other pieces of DNA.

Antibiotic selection

place 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 • Each entry has an accession number that scientists use to refer back to the cloned sequence • Maintained by the National Center for Biotechnology Information (NCBI) - NCBI is goldmine for bioinformatics resources that creates public access databases and develops computing tools for analyzing and sharing genome data

Library screening

rarely results in the cloning of the full length gene partly because this occurs with cDNA libraries - difficult to isolate full length mRNA or synthesize full-length cDNA for the gene of interest. Usually get small pieces of teh gene - look for overlapping sequences which can be pieced together to reform the full-length gene. Look for start and stop codons to know where the full length of the gene is obtained.

Chain termination sequencing (using original Sanger method)

reaction tube contains single stranded primer annealing to denatured DNA template, 4 types of dNTPs, DNA polymerase, and dideoxynucleotide (ddNTP). - ddNTP has a 3'H instead of 3'OH - when ddNTP incorporate into DNA chain, deoxyribose do not have OH to form phosphodiester bond with the dNTPs, so chain gets terminated.

low % gel (0.5%)

resolves larger size fragments

Agarose Gel Electrophoresis

separate and visualize DNA fragments based on size. Agarose is isolated from seaweed. It is melted in a buffer solution and poured into a horizontal tray. As it cools it will form a semisolid gel containing small pores through which DNA will travel. The persentage of agrose used to make the gel determines the ability of the gel to separate DNA fragments of different sizes

Future NGS

third generation sequencing using nanotechnology to push single strand nucleotide fragments into nanopores and then cleaving off individual bases to produce a signal that can be captured. This new approach will not involve DNA amplification or fluorescent tags so it is truly direct sequencing

RNA polymerase promoter sequences

used for transcription in vitro and in vivo. In vivo: allow bacterial cells to make RNA from cloned genes - synthesize proteins. In vitro: transcribed RNA can synthesize RNA probe - useful in studying gene expression

Bacteriophages (phages)

viruses that infect bacterial cells

When to do microarray

when compare two different conditions with 2 different colored dyes (one for treatment and other for control condition). Laser is scanned at different wavelengths for each probe and then the images are overlaid to make direct comparisons between the treatment and control • e.g. study gene expression difference between cancer cells and normal cells to look for genes possibly involved in cancer progression • Results of such studies can possibly lead to new drug therapies to combat cancer and other diseases

Aims of the HGP

• Analyze genetic variations among humans. This included the identification of single-nucleotide polymorphisms • Map and sequence the genomes of model organisms, including bacteria, yeast, roundworms, fruit flies, mice, and others • Develop new laboratory technologies such as high-powered automated sequencers and computing technologies, as well as widely available databases of genome information, which can be used to advance our analysis and understanding of gene structure and function • Disseminate genome information among scientists and the general public • Consider the ethical, legal, and social issues that accompany the HGP and genetic research