Chapter 20: Recombinant DNA Technology and Gene Cloning

Describe: Fluorescent in situ hybridization

(FISH) Involves hybridizing a probe directly to a chromosome or RNA without blotting Carried out with isolated chromosomes on a slide or in situ in tissue sections or entire organisms Helpful when embryos are used for various studies in developmental genetics

Describe: pUC18

~2600 base pair segment Allows for the insertion of large DNA Contains an origin of replication Can produce ~500 copies per cell Has a polylinker site = large # of RE sites LacZ gene marker

The number of clones within a library equals...

The certain probability of containing all the genomic sequences

Describe: Next-generation sequencing

Allows faster and cheaper genomic sequencing ~200 X faster

Describe: Plasmids

Extrachromosomal double stranded DNA molecule Replicates in bacterial cells Numerous restriction enzyme sites Contains a marker gene

Describe: Southern Blot

DNA Used to identify which clones in a library contain a given DNA sequence Used to characterize size of fragments from restriction digest Methodology: Hybridization between complementary nucleic acid (DNA or RNA) molecules

How are genomic libraries constructed?

DNA extracted from cells or tissues and cut with restriction enzymes and ligating the fragments into vectors

Describe: DNA Ligase

DNA fragments will seal phosphodiester backbone Joins restriction fragments covalently to produce intact DNA molecules

Describe: Probes

DNA or RNA Complementary to the gene of interest Screen library to recover clones DNA or RNA - labeled with radioactivity or fluorescent probe

Describe: Expression Vectors

Designed to ensure mRNA expression of cloned genes Produce large quantities of encoded protein within the host cell Transcription and translation of the gene itself is done by host machinery

Describe: DNA Sequencing

Determines the sequences of bases in DNA

Describe: Dideoxy Chain Termination Sequencing

Developed by Sanger Based on replication DNA fragment to be sequenced is converted to single stranded molecules and used as a template for synthesizing a series of complementary strands Each of the strands randomly terminates at a different specific nucleotide 1. DNA is denatured into single strands 2. DNA is mixed with primers that anneal to 3' end of DNA 3. DNA polymerase, dNTPs, and ddNTP added 4. As DNA is synthesized, ddNTP is inserted occasionally into the DNA, synthesis is terminated

Describe: Automated DNA Sequencing

Fluorescent dye is used attached to ddNTPs Gel is scanned with a laser Laser determines if A, C, T, or G was inserted Data is represented as colored peaks corresponding to 1 nucleotide

Describe: Selectable marker genes

Genes that provide resistance to antibiotics found on the plasmid

What are the two main types of DNA Libraries?

Genomic Complementary

Describe: DNA Libraries

Represent a collection of cloned DNA Two main types

Describe: Multiple cloning sites

Restriction sites for commonly used restriction enzymes allow scientists to clone a range of different fragments

Describe: RT-PCR

Reverse-Transcription PCR Used to study gene expression by studying mRNA production by cells or tissues More sensitive - can identify mRNA with low copies in the cell

Describe: Recombinant DNA Technology

There are a variety of molecular techniques Process of joining molecules from different sources Must isolate, replicate, and analyze genes

Describe: Blue-White Selection

Used to identify cells containing recombinant and nonrecombinant DNA Plasmid contains lacZ gene, which encodes beta-galactosidase Agar plates contain X-gal which is analogous to lactose, the substrate for beta-galactosidase When X-gal is cleaved by an enzyme it turns blue Bacterial cells with functional lacZ (carrying a nonrecombinant plasmid) will appear blue Bacterial cells with recombinant plasmids will appear white

Describe: Using Embryonic Stem Cells in Knockout Mice

Using ES cells, scientists introduce targeting vectors into cells via electroporation ES cell takes in targeting vector and endogenous enzyme recombinase catalyzes homologous recombination Recombinant ES cells are injected into mouse embryo Results: Chimeras

_______ depends on size of the genome.

Vector

Describe: Ti Vectors

Vectors for Plant Cells Rhizobium radiobacter Soil bacteria that infects plant cells and produces tumors Contains plasmid (Ti plasmid - tumor-inducing plasmid) Is used to transform plant cells with T-DNA containing foreign DNA Tumor-inducing gens can be removed from the vector so the recombinant vector does not produce tumors

What vector was utilized for the Human Genome Project?

YACs

Describe: YACs

Yeast Artificial Chromosomes Have telomeres at each end, ORI, and centromere 100-1000 kilobase inserts Important for the Human Genome Project

Describe: Saccharomyces cerevisiae

Yeast is widely used as host for DNA cloning and expression of eukaryotic genes Can be grown easily and manipulated Genetics have been studied intensively Entire genome has been sequenced Posttranscriptionally modify eukaryotic proteins Considered to be safe

Describe: Nucleic Acid Blotting

Southern: DNA Northern: RNA Western: Protein

Describe: cDNA Library

mRNA that is actively expressed in a cell Transcriptionally active genes No sequences adjacent to the gene

Describe: Applying a Probe

1. Colonies of the library are overlaid with a DNA-binding membrane 2. Colonies are transferred to membrane, then lysed, and DNA is denatured 3. Membrane is placed in a heat-sealed bag with a solution containing the probe; the probe hybridizes with denatured DNA from colonies 4. Membrane is rinsed to remove excess probe, the dried; X-ray film is placed over the filter for autoradiography 5. Using the original plate, cells are picked form the colony that hybridized to the probe 6. Cells are transferred to a medium for growth and further analysis

Describe: Creating Knockout Mice

1. Construct a targeting vector: Create a segment of DNA for introduction into a cell 2. Undergoes homologous recombination with gene of interest and renders it non-functional 3. Target vector has mutated copy of gene of interest 4. Foreign DNA disrupts reading frame and produces non-functional protein

Describe: Southern Blotting Technique

1. DNA is cut into fragments with restriction enzymes 2. Fragments are separated on a gel 3. DNA is denatured 4. DNA is transferred to the membrane 5. Membrane is incubated with radioactive probe 6. Hybridization of probe with gene of interest 7. Washed 8. Visualized on X-ray film 9. Compare original gel to radiolabeled autoradiograph

Describe: Third Generation Sequencing

1. DNA polymerase located in a nanopore anchored to a solid substrate binds a single-stranded DNA molecule to be sequenced. 2. DNA polymerase adds fluorescently tagged nucleotides to synthesize DNA 3. Fluorescent tag is cleaved off each base as it is added to the DNA strand. 4. Each light signal admitted is unique to each individual nucleotide Occurs in "real-time"

What are the three steps of PCR?

1. Denature (95 C) 2. Primer Annealing (50-70 C) 3. Extension(72 C)

What does nucleic acid blotting identify and characterize?

1. If the clone contains part of a gene 2. Ascertain the size 3. Organization

What are the important characteristics for vectors?

1. Must have Origin of Replication 2. Must have a selectable marker 3. Must have restriction enzyme sites

How is a gene inserted into a vector?

1. Plasmid vector is removed from bacterial cell and cut with a restriction enzyme 2. DNA to be cloned in cut with the same restriction enzyme 3. The two DNAs are ligated to form a recombinant molecule 4. Introduction into bacterial host cells by tranformation

What are the four types of vectors?

1. Plasmids 2. Lambda Phage 3. Bacterial Artificial Chromosomes (BACs) 4. Yeast Artificial Chromosomes (YACs)

Describe: Advantages of cDNA Library

1. Repetitive DNA not transcribed 2. Active Genes only 3. Introns are not contained

What are the reporter probes used in qPCR?

1. SYBR green 2. TaqMan

What are the two methods to recover transformed cells?

1. Selectable marker genes 2. Blue-White Selection

Describe: Disadvantages of cDNA Library

1. Sequences present only in mature mRNA 2. No promoter or enhancer sequences

Describe: PCR Limitations

1. Some information about nucleotide sequence of target DNA is required to synthesize primer 2. Minor contaminations from other sources can cause problems 3. PCR cannot amplify long segments of DNA 4. Taq Pol does not proofread

What are the two main techniques for transformation?

1. Using calcium ions and brief heat shock to pulse DNA into cells 2. Electroporation: A brief but high-intensity pulse of electricity to move DNA into bacterial cells

What is the formula for calculating the approximately size of a piece of DNA after cutting with a restriction enzyme?

4^n n = number of base pair recognition site

Describe: Vector

A carrier of a DNA molecule that replicates independently from the host chromosome Can replicate cloned DNA fragments in host cell Several restriction enzyme sites allow the insertion of the DNA fragment Selectable marker is required to identify these cells Must be easy to recover

What cells are used as hosts?

Bacteria Plants Insects Animals Yeast

Describe: BACs

Bacterial Artificial Chromosomes Generally very large Low copy number per cell 100-300 kilobase insert

Describe: Lambda Phage

Bacteriophage Central 1/3 of the vector is replaced with foreign DNA ~45 kilobase insert Introduced into bacteria by phage infection

What types of ends are formed by restriction enzymes?

Cohesive/Sticky Ends Blunt Ends

What is a genomic library?

Collection of clones containing all the DNA fragments from one source

What is cDNA?

Complementary DNA Complementary to mRNA

Describe: Genomic Library

Contains at least one copy of all sequences in a genome of interest Constructed by cutting genomic DNA with restriction enzyme and ligating fragments into vectors

Describe: Digestion Patterns of Fragments

Created by cutting DNA with different restriction enzymes and separating DNA fragments by gel electrophoresis Separated by size Fragments visualized with UV Transilluminator and ethidium bromide

Clones may contain:

Entire gene of interest Parts of the gene No part of the gene

Describe: Transgenic Animals

Express or overexpress a particular gene of interest (transgene) Vector with transgene undergoes homologous recombination into host genome Vector with transgene can also be put into ES cells and injected into embryos Allows for study of effects on appearance and function in mice

Describe: cDNA Library Construction

Isolating mRNA from cells Synthesizing complementary DNA using reverse transcriptases -Used to generate cDNA from mRNA -Extends oligo (dT) primer and synthesizes complementary DNA copy of mRNA -Produces mRNA-DNA double stranded hybrid molecule RNase H Pol I Ligase Cloning cDNA into vector

Describe: pUC18 and Blue-White Screening

LacZ+ colonies = blue in presence of X-gal LacZ- colonies = DNA fragment was inserted into the lacZ/polylinker region = colonies are white

Describe: Restriction Mapping

Number and order of restriction sites can be determined Distance between restriction sites can be determined Separated by gel electrophoresis Unites expressed in base pairs

Describe: Transformation

Plasmids are introduced into bacteria via transformation

Describe: PCR

Polymerase Chain Reaction Copies a specific DNA sequence through in vitro reactions Requires two oligo primers

Describe: Specific Clones Can Be Recovered from a Library

Probes are used to sort through a library and isolate specific genes of interest

Describe: Restriction Enzymes

Produced naturally by bacteria as a defense mechanism against bacteriophages Cleaves the DNA at specific recognition sequences known as pallindromes

Plasmids, phage vectors, and YACs only carry DNA and do not signal for a __________.

Protein

Describe: qPCR

Quantitative real-time PCR Allows researchers to quantify amplification reactions as they occur in real time

Describe: Northern Blotting

RNA that is actively being expressed in a given cell or tissue Used to study patterns of gene expression in embryonic tissues, cancer, and genetic disorders

How is recombinant DNA created?

The generation of specific DNA fragments Uses: Restriction Enzymes, a Vector to transfer the genetic material to a host cell, and utilizes the host cell machinery to produce many copies. The material must also be able to be recovered from the host cell

Describe: Gene Knockout

To disrupt or eliminate specific gene(s) and see "what happens" Knockout mice have revolutionized research

Describe: Gene Targeting

To manipulate a specific allele, locus, or base sequence and learn its function on a gene of interest Manipulates a specific gene within a genome

If a Ti Vector integrates into a plant chromosome it is said to be __________.

Transgenic