Chapter 20: Recombinant DNA Technology

Screening a library with a probe

*Get a probe that represents a gene you want to find 1. Bacterial clones from the library are grown on nutrient agar plates, forming thousands of colonies 2. Copies of the colonies are made using a nitrocellulose membrane 3. The membrane is processed to: -lyse the bacterial cells -denature the double-stranded DNA released from the cells into single strands -and bind these strands to the membrane 4. The DNA on the membrane is screened by incubation with a labeled nucleic acid probe a. The probe is heated and then cooled quickly to form single stranded molecules b. Then its added to a solution containing the membrane -If the nucleotide sequence of the DNA on the membrane is complementary to the probe, a double stranded DNA-DNA hybrid molecule will form (hybridization) 5. The remaining membrane is assayed using xray film to detect the remaining hybrid molecules -Radioactivity from the probe molecules bound to the DNA will expose dark spots on the film -spots are colonies containing the cloned gene of interest

Key properties of DNA vectors

-A vector contains several restriction sites that allow insertion of the DNA fragments to be cloned -Vectors must be capable of replicating in host cells -To allow for independent replication of the vector DNA and any DNA fragment it carries -Vector should carry a selectable marker gene (usually and antibiotic resistance gene or gene that encodes a protein which produces light) -In order to distinguish host cells that have taken up vectors from those that have not Many vectors incorporate specific sequences that allow for sequencing inserted DNA

PCR cloning advantages over DNA cloning

-PCR is rapid -PCR is sensitive: amplifies specific DNA sequences from small DNA samples (even DNA in a single cell) PCR was used in the HGP

Read Length

-The amount of sequence that can be generated in a single individual reaction AND -The total amount of DNA sequence generated in a sequence run

Restriction sites

-are present randomly in the genome -the number of DNA restriction fragments produced by digesting DNA with a particular enzyme can be estimated from the number of times a given restriction enzyme cuts the DNA -i.e. an enzyme with 4 base recognition sequence will cut every 4^n (4^4) = 265 base pairs

BACs and YACs

-are vectors that can be used to clone large fragments of DNA BACs: -bacterial artificial chromosomes -are very large, but have a low copy number -take up large fragments, but smaller than the fragments YAC takes up -used in the HGP YACs: -yeast artificial chromosomes -contain: telomeres, ori's, and centromere (are linear) - can clone DNA inserts from 100-1000 kb (up to 2 megabases of DNA) -also used in the HGP -YACs are separated using large pulsed field gel electrophoresis (the largest chromosome is at the top) (but read from the bottom)

Cloning Methods

-embryo splitting -cell fusion -nuclear transfer (Dolly the sheep)

Restriction enzymes

-proteins that are molecular knives: -cut, cleave, and digest DNA at specific recognition sequences (specific locations of the genome) Discovered in bacteria as a defense mechanisms (against infection by bacteriophage) -they bind to the viral DNA -scan to find the recognition sequence -then cut the viral DNA into pieces, peeving infection -they recognize and bind to DNA at specific nucleotide sequences, call recognition sequences or restriction sites -then, it cuts both strands of DNA within that sequence -cleaves the phosphodiester backbone of DNA ("digestion")

Limitations of PCR

-some info about the nucleotide sequence of the target DNA must be know, in order to synthesize primers -minor contaminations of the DNA sample will cause problems (i.e. skin cell from the researcher) -PCR must always be performed in parallel with carefully designed controls -Typically, PCR cannot amplify long segments of DNA

DNA Sequence Analyzer

-synthesizes DNA -separates fragments -analyzes results

Limitations of plasmids

-they are small, so can only accept inserted pieces of DNA that are up to ~25 kb

Using PCR to clone DNA

1. A double stranded target DNA (the one to be cloned) is placed in a tube with: -DNA polymerase -Mg2+ -four deoxyribonucleoside triphosphates (dNTP) 2. A sample of DNA is denatured into single strands 3. Sequence info (about the target DNA) is used to synthesize two oligonucleotide primers (a short, single stranded DNA sequence) -One primer that is complementary to the 5' end of one strand of target DNA -Another primer complementary to the opposing strand of target DNA (3' end) 4. The primers are added to the sample of denatured DNA, in which they bind to complementary nucleotides 5. Polymerase extends the 3' ends of each primer to synthesize second strands of the target DNA

Phage vector cloning steps

1. DNA fragments are ligated into the phage vector to produce recombinant lambda vectors 2. the lambda vectors are packaged into phage protein heads in vitro and introduced into the bacteria host cells growing on petri dishes 3. Inside the bacteria, the vectors replicate and form many copies of infective phage -each copy carries a DNA insert 4. As they reproduce, they lyse their bacterial host cells, forming clear spots called plaques -the phage can be isolated from the plaques and the cloned DNA can be removed

Creating a Southern blot

1. DNA is cut into fragments 2. fragments are separated by gel electrophoresis 3. DNA in the gel is denatured into single strands (to prepare for hybridization) 4. DNA fragments are transferred from the gel to the membrane substrate 5. Membrane is labeled with a single-stranded DNA probe and hybridized 6. The DNA fragments (on the membrane) that are complementary to the probes nucleotide sequence bind to the probe and form double-stranded hybrids 7. The excess probe is washed off 8. Membrane is exposed to X-ray film 9. Film is developed and pattern is analyzed

Making a genomic library

1. DNA is extracted from cells or tissues and cut with restriction enzymes 2. The resulting DNA fragments are inserted into vectors -it is important to chose a vector that contains the entire genome in the smallest number of clones

Routine PCR 3-step Cycles

1. Denaturation: -the double stranded DNA to be cloned is denatured into single strands (heated @ 92-95 degrees C for ~1 min) 2. Hybridization/Annealing: -the temp. is lowered to 45-60 degrees C -causes hybridization/annealing (primer binding) to the denatured, single stranded DNA region 3. Extension (DNA synthesis): -temp. adjusted to 65-75 degrees C -DNAP uses the primers as a starting point (template) to synthesize new DNA strands -adds nucleotides to the ends of the primers in from 5' --> 3' -repeated cycles of PCR can amplify the original DNA sequence by more than a millionfold

Creating a cDNA library

1. Isolate mRNA from a population of cells of interest -this mature mRNA has already been processed (w/ a tail and cap) 2. mix mRNAs with oligo (dT) primers -oligo primers are short, single stranded sequences of T nucleotides that anneal to the poly-A tail of the mRNA 3. Reverse transcriptase extends the oligo (dT) primer and synthesizes a complementary DNA copy of the mRNA sequence -product = an mRNA-DNA double stranded hybrid molecule -the mRNA is digested by RNAse H, producing gaps in the RNA strand 4. A second, opposing strand of DNA is synthesized by DNAP -overtime, all the RNA in the hybrid will be replaced w/ DNA -this creates double stranded cDNA molecules that are complementary to the mRNA 5. the cDNA molecules are subsequently inserted into vectors (usually plasmids) -linker sequences have to be attached to the ends of the cDNA (in order to know what restriction enzyme to use) 6. Linker sequences are cut with EcoRI and ligated to vectors treated with the same enzyme 7. The vectors carrying cDNA molecules are transferred to host cells and cloning is used to make a cDNA library

Plasmid Components

1. Origin of replicate site (ori): -where replication begins -found in every bacterial genome -they can increase their copy number to produce hundreds of copies in a single host cell 2. Multiple cloning site (MCS): -a.k.a the polylinker region -the region that contains multiple restriction target sites -important because this is how your gene of interest is inserted into the plasmid -allows scientists to clone a range of different fragments generated by many commonly used restriction enzymes 3. Selection mechanism: -a way for the plasmid to select bacteria that are resistant to it -usually an antibiotic resistance gene/sequence i.e. antibiotic resistance gene i.e. blue-white selection -based on the expression of X gal gene (pg 493)

Dyes used in qPCR

1. SYBR green -binds to double stranded DNA 2. TaqMan Probes -are complementary to specific regions of the target DNA, between where the forward and reverse primers for PCR bind to -contain two dyes: a. The Reporter: -located at the probe 5' end -releases fluorescent light when excited b. The Quencher: -attached to the probe 3' end

Components to Southern blotting

1. Separation of DNA fragments by gel electrophoresis -used to characterize the number of fragments produced by restriction digestion -also to estimate the molecular weights of the fragments 2. Hybridization of the fragments using labeled probes -hybridization characterizes the DNA sequences present in the fragments, so they are identified!

Steps for cloning DNA using plasmid vectors

1. The plasmid DNA and DNA to be cloned are cut using the same restriction enzyme -the plasmid is cut once within the multiple cloning site to produce a linear vector -DNA restriction fragments from the DNA to be cloned are added to the linearized vector in the presence of DNA ligase 2. Sticky ends of DNA fragments anneal, joining the DNA to be cloned and the plasmid 3. DNA ligase creates phosphodiester bonds to seal nicks in the DNA back bone -thus, producing recombinant DNA 4. Once inside the cell, plasmids replicate quickly to produce multiple copies

Transformation techniques

1. Treat cells with calcium ions and use brief heat shock to pulse DNA into cells 2. Electroporation -uses brief, but high-intensity, pulse of electricity to move DNA into bacterial cels

Advantage of cDNA library

1. cDNA is complementary to the nucleotide sequence of mRNA -so it contains only expressed (coding, not non-coding) genes, unlike genomic libraries 2. cDNA libraries provide a snapshot of the genes that were transcriptionally active in a tissue at a particular time because the relative amount of cDNA in a particular library is equal to the amount of starting mRNA isolated from the tissue and used to make the library

Solid phase method

A method of NGS created by Roche 454 Process: 1. Beads are attached to fragmented DNA 2. Each bead is amplified with PCR in separate water droplets in oil 3. Beads are loaded onto multi-well plates and mixed with DNAP 4. Pyrosequencing is used to sequence DNA on the beads in each well -a single nucleotide is flowed over the well -when a complementary nucleotide crosses a template strand adjacent to the primer, it is added to the 3' end of the primer -this addition initiates a chemiluminescent reaction that produces light 5. Emitted light is recorded to determine when a single nucleotide has been incorporated into a strand

Clone

A molecule, cell, or individuals derived from a single ancestor Examples: -MZ twins -crop plants -some types of trees -Dolly the sheep

Probes

A probe is any DNA/RNA sequence that is complementary to some part of a cloned sequence in the library, and labeled with fluorescence Probes are tagged in different ways so they can be identified -i.e. using radioactive isotopes or non-radioactive compounds that undergo chemical or color reactions, which indicate the location of a specific clone in a library -when used in a hybridization reaction, the probe binds to any complementary DNA sequences on the chromosome, present in more than one clone Deriving probes -they are derived from many sources, but often related to genes isolated from another species

Power of DNA technology

Allows researchers to identify and isolate a single gene DNA segment of interest from a genome Use of cloning to produce large quantities of copies -these clones can then be manipulated for many purposes: -research on the structure and organization of DNA -studying gene expression -studying protein products -producing commercial product from protein encoded by a gene

Hybridization

Base pairing single stranded DNA/RNA molecules to their complementary DNA/RNA

Cloning vectors

Cloning vectors are DNA molecules that accept DNA fragments and replicate them when vectors are introduced into host cells (vectors are carrier DNA molecules) -A vector combine with a DNA restriction fragment forms a recombinant DNA molecule -vectors differ in terms of the host cells they are able to enter and in the size of the DNA fragment inserts they can carry

Building DNA Microarrays

Components needed: -Substrate: glass, silicon chip -Single stranded DNA attached as a probe -Single stranded target DNA/RNA labeled with fluorescent dye

Computer-automated high-throughput DNA sequencing

Computer automated Sanger-based sequencing -generates large amounts of sequence DNA

Recombinant DNA

DNA created by artificially joining pieces of DNA from different sources Tools needed to construct and amplify recombinant DNA: 1. restriction enzymes are used to cleave DNA at specific sequences 2. DNA cloning vectors

Cleavage patterns

DNA is cut in a characteristic cleavage pattern: -most common are four or six, but can be eight nucleotides long 1. Cohesive ends ("sticky ends") -fragments with single stranded overhanging ends produced by offset cuts -i.e. enzymes EcoRI and HindIII -cohesive ends make cloning less challenging because they are facilitated by hydrogen bonding 2. Blunt ends -strands cut at the same nucleotide pair, creating fragments with double stranded ends -i.e. enzymes AluI and BalI

Expression vectors

Designed to ensure mRNA expression of the cloned gene, with the purpose of producing many copies of the gene's encoded protein in a host cell -used in both prokaryotic and eukaryotic host cells

DNA Microarrays pgs. 646-651

Diagnosis can be made by screening all genes in the genome at once -this method uses DNA microarrays, a.k.a GeneChips Goal for typical experiment: -identify genes expressed in cancer cells, but not in normal cells -traditionally, experiments had to be done one at a time to identify these genes -DNA microarrays can identify hundreds of these genes in a single experiment -you can attach DNA/RNA nucleotides onto it as a substrate and immobilize it -then you to a hybridization experiment Gene expression profiles can be used to diagnose cancer or to differentiate one cancer from another

Sanger Sequencing

Dideoxynucleotide chain-termination sequencing -the most common method of DNA sequencing and the basis for other sequencing technologies -developed by Sanger Components needed: -DNA template -primers -DNAP -dNTPs with chloroforms (fluorescent molecules) Process: 1. A double stranded DNA molecule, (whose sequence is to be determined) is converted to single strands 2. the single strands are used as a template for synthesizing a series of complementary strands 3. the DNA to be sequenced is mixed with a primer/vector 4. The four deoxyribonucleotide triphosphates are added to each tube -dATP, dCTP, dGTP, dTTP

Recognition sequences

Exhibit a form of symmetry called palindrome -the nucleotide sequence reads the same on both strands of DNA when read in the 5' to 3' direction

FISH

Fluorescent in situ hybridization (FISH) -involves hybridizing a probe directly to a chromosome or RNA without blotting -carried out with isolated chromosomes on slide/in situ in tissue sections/entire organisms -a probe for a particular sequence is labeled with nucleotides that are tagged with a specific dye, which will fluoresce under fluorescent light -helpful when organisms are used for various studies in developmental genetics

Chromosome specific library

Grow cells from an organism If you isolate cells in metaphase, you can see them as distinct chromosomes (stained with G/C and A/T fluorescent dyes) -the chromosomes are put through a sorter and a laser segregates the colors -DNA is extracted and cloned into a vector

Genomic Libraries

Ideally, a genomic library consists of many overlapping fragments of the genome, with at least one copy of every DNA sequence in an organisms genome -in summary, it spans the entire genome Genomic libraries contain coding and non-coding segments of DNA (introns) Slowly being replaced with whole-genome shot-gun cloning

Phage vectors

Includes genetically modified strains of lambda-phage: -lambda-phage is a virus that infects E.coli -its genome has been modified to incorporate many of the necessary features that cloning vectors need -including multiple cloning sites -phage vectors can carry up to 45 kb size DNA fragments

Ti Vectors

Insertion of genes into plant cells using the soil bacterium: Rhizobium radiobacter -infects plant cells and produces tumors (crown galls) Rhizobium contains a plasmid called the Ti plasmid (tumor-inducing) -genes in the T-DNA segment control tumor formation and synthesis of compounds required for growth of the infecting bacteria Restriction sites in Ti plasmids are used to insert foreign DNA -once inside the cell, the foreign DNA is inserted into the plant genome when the T-DNA integrates into a host cell chromosome Plant cells carrying recombinant Ti plasmid can be grown in tissue or culture -the presence of certain compounds in the culture medium stimulates the formation of roots and shoots

Restriction Mapping steps overview

Involves: 1. Digesting DNA with one or more restriction enzymes -Single digest (one enzyme at a time) -Double digest (two enzymes at a time) 2. Running DNA fragments on a gel via electrophoresis 3. Analyzing patterns of fragments in each lane 4. Drawing a physical map that will agree with the fragments produced by every enzyme in the experiment

Linker sequences

Linker sequences are short, double-stranded oligonucleotides containing a restriction enzyme recognition sequence

Whole-genome shot-gun cloning

Method that allows you to sequence all of the DNA fragments in a genomic DNA sample, without the need for inserting DNA fragments into vectors and cloning them into host cells -This method is slowly replacing traditional genomic libraries

Modifications of the Sanger technique

Modifications allow sequencing rxns to occur in a single tube, in which all four ddNTPs are labeled with a different colored fluorescent dye Process: 1. As DNA fragments move through the gel, they are scanned with a laser 3. The laser stimulates the fluorescent dyes on each DNA fragment, which emit different wavelengths of light for each ddNTP 3. The emitted light is captured by a detector that amplifies and feeds info into a computer 4. The computer converts the light patterns into a DNA sequence

Next Generation Sequencing (NGS) technology

NGS technologies have the capacity to generate massive amounts of DNA for cheap Many companies are producing NGS: 1. Applied Biosystems (ABI): -created SOLiD: supported oligonucleotide ligation and detection -can produce 6 gigabases of sequence data per run 2. Roche 454 Life Sciences: -first company to commercialize NGS -sequences genomes using a solid phase method -generates long reading lengths 3. Helicos Biosciences 4. Illumina: -method that attaches DNA fragments to a solid support and then uses sequencing rxns (that are similar to Sanger) -this generates shorter reading lengths, but the number of sequencing reads is higher

EcoRI

One of the first restriction enzymes to be identified was isolated from EcoRI DNA fragments produced by EcoRI digestion have cohesive ends -they can base pair with complementary single-stranded ends on other DNA fragments cut using EcoRI -when mixed together, single stranded ends of DNA fragments from different coerces cut with the same restriction enzyme can anneal -the addition of DNA ligase to DNA fragments will seal the phsophodiester backbone of DNA to covalently join the fragments together to form recombinant DNA molecules

Key to the Sanger technique

Overview: ddNTP causes DNA synthesis to STOP because the polymerase does not recognize it The addition of a small amount of one dideoxynucleotide (ddNTP) -this is a modified deoxyribonucleotide: has a 3'-H instead of a 3'-OH (hydroxyl) ddNTPs are chain termination nucleotides: -they lack the 3' oxygen that is needed to form a phosphodiester bond with another nucleotide -because DNAP cannot add new nucleotides to the ddNTP, DNA synthesis is terminated this technique allows you to detect the ends of every fragment

PCR overview

PCR is a chain reaction -the number of new DNA strands is double in each cycle -the new strands and old strands served as templates in the next cycle -cycles take ~ 2-5 min and can be repeated immediately -the process is automated by therocyclers that can be pre- programmed to carry out a predetermined # of cycles -most PCR experiments have to go through ~40 cycles

Polymerase Chain Reaction (PCR)

PCR is a rapid method of DNA cloning that extends the power of recombinant DNA research -in most cases, it eliminates the need to use host cells for cloning PCR was invented in 1983 by Kerry Mullins PCR copies a specific DNA sequence through a series of in vitro reactions -these rxns amplify target DNA sequences that are initially present in small quantities Some information about the nucleotide sequence of the target DNA (of interest) is required One complete PCR process is called a cycle -one cycle doubles the number of DNA molecules in the reaction

Applications of PCR

PCR is one of the most versatile techniques, can be used for: -Screening mutations involved in genetic disorders -Diagnosing genetic disorders -Diagnosing methodology for the detection of bacteria and viruses in humans and pathogenic bacteria in food -Studying samples from singles cells, fossils, or even crime scenes -used in human IDs Important PCR techniques: 1. Reverse transcription PCR (RT-PCR) 2. Quantitative real-time PCR (qPCR)

Bacterial plasmid vectors

Plasmids-extrachromosomal double stranded DNA molecules that replicate independently from the chromosome within bacteria cells (they are circular) Genetically modified bacterial plasmids were the first vectors developed and are still widely used for cloning Plasmids are made up of 3 components: 1. Origin of replication site (ori) 2. Multiple cloning site (MCS) 3. Selection mechanism -plasmids must be linearized in order to add the gene Plasmids are introduced into bacteria through transformation -only one or a few plasmids enter a host cell

Reverse transcription PCR (RT-PCR)

RT-PCR is used for studying gene expression -mRNA production by cells or tissues Procedure: 1. RNA is isolated from cells or tissues to be studied 2. Reverse transcriptase is used to generate double stranded cDNA molecules 3. The rxn is then followed by PCR to amplify cDNA 4. Amplified cDNA fragments are separated onto agarose gel and inserted into plasmid vectors

DNA Libraries

Represent a collection of cloned DNA samples derived from a single source that could be a particular tissue type, cell type, or single individual There are two types of libraries: 1. Genomic DNA libraries 2. Complementary DNA (cDNA) libraries

Restriction Mapping

Restriction enzymes were used to make the original physical maps -you can cut DNA and run the fragments on gels, then analyze them -fragments are separated by size and then are put back together on the map Restriction maps establish the number of, order of, and distances between restriction enzyme cleavage sites along a cloned segment of DNA -provides info about the length of the cloned insert (fragment) -and the location of restriction enzyme cleavage sites within the clone -this data can be further used to re-clone fragments of a gene and compare its internal organization with that of other cloned sequences Because of bioinformatics, maps are now created using software -software identifies the cleavage sites in the sequence DNA

Sequencing technologies

Sanger is becoming outdated: -it is expensive -the output is not high enough to support the demand for genomic data Next generation sequencing (NGS) technology -allows for the development of sequencing a complete individual genome for $1000

Screening

Screening is the process of recovering genes from a library -to find a specific gene in the library, we need to identify and isolate only the clone or clones containing that gene Library screening methods: -Probes: any DNA/RNA sequence that is complementary to some part of a cloned sequene present in the library (the target sequence)

Selectable marker genes

Selectable gene markers provide resistance to antibiotics -i.e. ampicillin, lacZ gene Used to identify bacteria containing recombinant plasmids, a.k.a "blue-white" selection -a plasmid is used that contains the lacZ gene incorporated into the multiple cloning site -the lac Z encodes for beta-galactosidase -blue white selection takes advantage of the enzymatic activity -if a DNA fragment is inserted anywhere in the multiple cloning site, the lacZ gene is disrupted and will not produce functional copies of beta-galactosidase X-gal is similar to lactose -When cleaved by beta-galactosidase, it turns blue -Recombinant bacteria with palsmids containing an inserted DNA fragment form white colonies on X gal -Bacteria in the white colonies are clones of each other

Other Blotting approaches

Southern Blotting led to the development of other blotting approaches: 1. Northern blot analysis: -RNA blotting 2. Western blotting: -used to analyze proteins 3. FISH: -fluorescent in situ hybridization 4. Spectral Karyotypes: -variation of the FISH technique to produce karyotpes -individual chromosomes can be detected using probes labeled with dyes that that fluoresce at different wavelengths -this is valuable in detecting deletions, translocations, duplications, and other chromosome structure anomalies

Nucleic Acid Blotting

Southern blotting is a method used to detect hybrids -a way to transfer DNA from a gel to filter paper Used to: 1. Identify which clones in a library contain a given DNA sequence 2. characterize the size of the fragments 3. identify fragments carrying specific genes in genomic DNA that was digested -these fragments are isolated & re-cloned 4. Identify the number of copies of a particular sequence/gene that are present in a genome

DNAP used in PCR

The DNA polymerase used in PCR needs to be thermally stable -because of the multiple heating and coolings Taq Polymerase -derived from the hot springs of Yellowstone -an enzyme capable of tolerating extreme temperature changes

Recombinant DNA technology

The methods used to copy or clone DNA -Often known as "gene splicing"

Molecular Techniques for Analyzing DNA

These are methods that provide info about the organization and function of the cloned sequences 2 Techniques: 1. Restriction Mapping 2. Nucleic Acid Blotting

Problems that can arise with cloning DNA using plasmid vectors

When cloning DNA using plasmids, not all plasmids will incorporate DNA -using a particular restriction enzyme, a plasmid can self-ligate (close back on itself) Not all host cells will take up plasmids -that's why it is important to use selectable marker genes

Host cells for cloning vectors

Which host cells are used to accept recombinant DNA for cloning? 1. E.coli used as a prokaryotic host cell when working with plasmids 2. Yeast Saccharomyces cerevisiae used as a host cell for the cloning and expression of eukaryotic genes - Yeast can be grown and manipulated in much the same way as bacterial cells -The genetics of yeast has been intensively studies, providing a large catalog of mutants and a highly developed genetic map -The entire yeast genome has been sequenced and genes in the organism have been identified -To study the function of some eukaryotic proteins, it is necessary to use a host cell that can modify the protein -for example by adding carbohydrates in order to convert it into functional form (bacteria cannot carry this out) -Yeast is a safe organism for producing proteins for vaccines and therapeutic agents

Complementary DNA (cDNA) library

cDNA libraries offer certain advantages over genomic libraries: They contain DNA copies, called cDNA -cDNA are made from the mature mRNA molecules of a cell population -the ones that have already been processed, so they have a tail and a cap -cDNA is complementary to the nucleotide sequence of mRNA -so it contains only expressed (coding, not non-coding) genes -cDNA libraries can be used to study certain cells or tissues under certain conditions -can also be used to compare expressed genes from normal tissues vs. diseased tissues

Quantitative real-time PCT (qPCR)

qPCR is one of the most valuable PCR techniques -allows us to determine the amount of PCR product created -enables researchers to quantify amplification rxns as they occur in real time Basic qPCR procedure: 1. Thermocyclers have a laser that scans a beam of light through the top or bottom of each PCR tube 2. Each rxn tube contains a probe or DNA-binding dye that emits fluorescent light when illuminated by the laser -the light emitted correlates to the amount of PCR product amplified

Anneal

stick together by hydrogen bonding of complementary base pairs in single stranded ends