Chapter 20: Biotechnology 1. DNA Sequencing 2. DNA Cloning 3. Studying Gene Expression 4. Manipulating Genomes 5. herapeutic & Diagnostic echniques
1. DNA Sequencing Chapter Reading pp. 409-412
DNA Sequencing ECHNIQUE DNA (template strand) C G A C C G A C A A Primer Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) G DNA polymerase dap dcp dp dgp P P P P P P G OH ddap ddcp ddp ddgp H G DNA replication in vitro using 1 of 4 different chainterminating dideoxynucleotides (ddnps) results in a set of DNA fragments ending in all positions with A, C, G, or that can be resolve by length on a gel
DNA Sequencing uses Chain erminators Normal nucleotide (dnp) Dideoxynucleotide (ddnp) *
DNA synthesis is carried out in reactions containing the following: DNA template to be sequenced dnp s DNA primer DNA polymerase ddap, ddcp, ddgp or ddp each ddnp is labeled with a different fluorescent tag he color reveals the identity of the ddnp that terminated DNA synthesis at each position, thus revealing the sequence!
DNA Sequencing ECHNIQUE (continued) C G A C C G A C A A DNA (template strand) ddc G Shortest Direction of movement of strands ddg C G dda G C G Labeled strands dda A G C G ddg G AA C G dd G G AA C G Longest labeled strand ddc G dda G C ddg A G C G AA G AA G AA C C C G G G Longest Detector Laser Shortest labeled strand
DNA Sequencing Direction of movement of strands Longest labeled strand Detector RESULS Laser Last nucleotide of longest labeled strand Last nucleotide of shortest labeled strand Shortest labeled strand G A C G A A G C
Next Generation Sequencing
2. DNA Cloning Chapter Reading pp. 409-417
What is Recombinant DNA? he joining of DNA from different sources. his can happen in nature (in vivo) the transfer of DNA involving bacteria or viruses or in the laboratory (in vitro) the cutting & splicing of DNA fragments by molecular biologists he term recombinant DNA generally refers to the in vitro kind which is commonly called gene cloning
Bacterium Gene Cloning 1 Gene inserted into plasmid Cell containing gene of interest Bacterial chromosome Plasmid Recombinant DNA (plasmid) 2 Gene of interest Plasmid put into bacterial cell DNA of chromosome ( foreign DNA) Recombinant bacterium gene of interest is inserted into a plasmid vector bacterial host transformed with recombinant plasmid bacterial clone with recombinant plasmid is identified
Gene Cloning Cloned gene can then be used in a variety of ways. 3 Host cell grown in culture to form a clone of cells containing the cloned gene of interest Gene of interest Protein expressed from gene of interest Copies of gene Protein harvested Basic research on gene 4 Basic research and various applications Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hormone treats stunted growth
1 2 3 DNA Restriction enzyme cuts sugar-phosphate backbones. DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. Restriction site G AA C G AA C C AA G C AA G One possible combination DNA ligase seals strands GAAC CAAG Sticky end Recombinant DNA molecule Restriction Enzymes Gene cloning usually involves the use of restriction enzymes that cut DNA at very specific sequences: e.g. EcoRI cuts at:..gaac....caag.. here are many different restriction enzymes, each cutting a different sequence
he Gene Cloning echnique ECHNIQUE Bacterial plasmid amp R gene lacz gene Restriction site Hummingbird cell cut plasmid & DNA to be cloned with same RE ligate vector & DNA insert with DNA ligase Sticky ends Recombinant plasmids Gene of interest Hummingbird DNA fragments Nonrecombinant plasmid transform bacteria with ligated DNA Bacteria carrying plasmids
Selection for Bacterial Clones Antibiotic resistance allows selection for bacterial clones containing the plasmid vector. RESULS Colony carrying nonrecombinant plasmid with intact lacz gene Bacteria carrying plasmids Colony carrying recombinant plasmid with disrupted lacz gene One of many bacterial clones Blue/white selection via lacz gene disruption allows identification of clones containing a DNA insert.
ECHNIQUE 1 Mixture of DNA molecules of different sizes 2 RESULS Longer molecules Power source Cathode Anode + Wells Gel Power source + Shorter molecules Gel Electrophoresis Separation of DNA fragments through a porous gel matrix: gel is either agarose or polyacrylamide electric current pulls negatively charged DNA toward the positive pole rate of movement is inversely proportional to DNA fragment size
DNA Libraries A collection of cloned genes from an organism is called a DNA library. Genomic DNA Library a collection of chromosomal DNA fragments cloned into a particular vector essentially cloned pieces of the organism s genome cdna Library a collection of DNA fragments produced from messenger RNA (mrna) cloned into a vector produced from mrna by reverse transcriptase a collection of expressed genes with NO intron DNA
DNA Libraries Foreign genome Cut with restriction enzymes into either small fragments or large fragments Bacterial artificial chromosome (BAC) Large insert with many genes Recombinant plasmids (b) BAC clone Plasmid clone (a) Plasmid library (c) Storing genome libraries
mrna DNA polymerase Reverse transcriptase cdna DNA strand DNA in nucleus mrnas in cytoplasm Poly-A tail Primer A A A A A A A A A A A A Producing cdna cdna (DNA complementary to mrna) is produced as follows: purify mrna fr. desired cell type treat with reverse transcriptase and oligo-d primer results in double-stranded DNA copies of all mrna from the cell clone cdna fragments into vector such as a plasmid transform bacterial hosts Results in a collection of cloned cdna corresponding to coding sequences of all expressed proteins
Screening DNA Libraries ECHNIQUE Multiwell plates holding library clones Radioactively labeled probe molecules Gene of interest Probe DNA Singlestranded DNA from cell CCACACCGGC GAGAGGGCCG Film Nylon membrane Location of DNA with the complementary sequence Nylon membrane DNA from library clones immobilized on a membrane radioactively labeled DNA similar in sequence to desired clone is added as a probe hybridization of probe to complementary DNA IDs clone
PCR ECHNIQUE arget sequence he Polymerase Chain Reaction is a technique to selectively amplify a desired DNA sequence: essentially DNA replication in vitro results in exponential amplification of target DNA sequence artificial primers specific for target DNA sequence limit replication to DNA complementary to primers Cycle 1 yields 2 molecules Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Genomic DNA 1 Denaturation 2 Annealing 3 Extension Primers New nucleotides
Overview of PCR Every PCR reaction requires the following: 1) source of target DNA template 2) artificial primers flanking DNA of interest 3) heat-stable DNA polymerase (from hyperthermophile) 4) dnp s 5) automated thermocycler to facilitate repeated: denaturation of DNA (separating the 2 strands) hybridization of primers to template DNA synthesis Resulting PCR products can then be analyzed by gel electrophoresis.
PCR Cloning involves designing restriction sites into the primers to facilitate cloning
3. Studying Gene Expression Chapter Reading pp. 417-419, 421-422
ECHNIQUE 1 cdna synthesis 2 PCR amplification 3 Gel electrophoresis RESULS Primers mrnas β-globin gene cdnas Embryonic stages 1 2 3 4 5 6 R-PCR produce cdna with reverse transcriptase carry out PCR using specific primers for desired gene gel electrophoresis of resulting PCR fragments Amount of DNA reflects amount of original mrna sequence.
in situ Hybridization
in situ Hybridization How is in situ hybridization carried out? fluorescently labeled anti-sense RNA probes added to tissue sample hybridization with complementary mrna sequences location of specific gene expression within the tissue (i.e., in situ) is revealed
DNA Microarrays A DNA microarray is a solid surface containing a precise array of single-stranded DNA sequences from 1000s of different genes in an organism. labeled cdna is produced from test cells and allowed to hybridize with sequences in the array intensity of signals reveal expression of specific genes within the test cells. When cdna from different sources is labeled differently, gene expression from each source can be compared in a single microarray (as shown on the slide after next).
ECHNIQUE 1 Isolate mrna. issue sample 2 Make cdna by reverse transcription, using fluorescently labeled nucleotides. mrna molecules Analyzing gene expression using a DNA microarray. 3 Apply the cdna mixture to a microarray, a different gene in each spot. he cdna hybridizes with any complementary DNA on the microarray. Labeled cdna molecules (single strands) DNA microarray DNA fragments representing a specific gene 4 Rinse off excess cdna; scan microarray for fluorescence. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample. DNA microarray with 2,400 human genes
Comparing Gene Expression
DNA Single Nucleotide Polymorphisms (SNPs) SNPs are single nucleotide differences that correspond with specific disease-causing alleles. PCR and hybridization techniques (e.g., microarrays) can reveal the presence of such alleles (genetic testing). SNP Normal allele C Disease-causing allele
4. Manipulating Genomes Chapter Reading pp. 422-425, 429-430
Cloning Plants Some plants can be cloned from a single adult plant cell. Cross section of carrot root 2-mg fragments Fragments were cultured in nutrient medium; stirring caused single cells to shear off into the liquid. Single cells free in suspension began to divide. Embryonic plant developed from a cultured single cell. Plantlet was cultured on agar medium. Later it was planted in soil. Adult plant
EXPERIMEN Frog embryo Frog egg cell Frog tadpole his experiment produced the first artificially cloned animal. Less differentiated cell Donor nucleus transplanted UV Enucleated egg cell Egg with donor nucleus activated to begin development Fully differentiated (intestinal) cell Donor nucleus transplanted RESULS Cloning Animals Most develop into tadpoles. Most stop developing before tadpole stage.
ECHNIQUE Mammary cell donor 4 5 6 RESULS 1 Cultured mammary cells Grown in culture 3 Implanted in uterus of a third sheep Embryonic development Egg cell from ovary Cells fused 2 Nucleus removed Nucleus from mammary cell Early embryo Surrogate mother Egg cell donor Lamb ( Dolly ) genetically identical to mammary cell donor he first cloned mammal, Dolly the sheep, was cloned by this method. Other mammals such as cats have since been cloned and presumably humans could be cloned this way as well.
ransgenic Organisms ransgenic organisms have a foreign gene (e.g. from another species) inserted into their genome. also known as genetically modified organisms (GMOs)
CRISPR-Cas9 a new method to modify genomes Cas9 active sites Guide RNA complementary sequence NUCLEUS Cas9 protein Guide RNA engineered to guide the Cas9 protein to a target gene 2 Part of the target gene Active sites that can cut DNA Cas9 guide RNA complex Complementary sequence that can bind to a target gene Resulting cut in target gene by Cas9 1 3 Normal (functional) gene for use as a template by repair enzymes CYOPLASM (a) Scientists can disable ( knock out ) the target gene to study its normal function. (b) If the target gene has a mutation, it can be repaired. NUCLEUS Random nucleotides Normal nucleotides
5. herapeutic & Diagnostic echniques Chapter Reading pp. 425-431
Stem Cells Embryonic stem cells Cells generating all embryonic cell types Adult stem cells Cells generating some cell types Stem cells are undifferentiated cells that can give rise to multiple differentiated cell types. Cultured stem cells Different culture conditions Different types of differentiated cells Liver cells Nerve cells Blood cells Embryonic stem cells are considered more pluripotent than adult stem cells, though techniques are being developed to expand the potential of adult stem cell.
Stem Cell herapy echniques are being developed to treat a person s own cells in vitro to produce a needed cell type which is then reintroduce into the patient to repair damaged tissue. 1 Remove skin cells from patient. 2 4 Patient with damaged heart tissue or other disease Return cells to patient, where they can repair damaged tissue. Reprogram skin cells so the cells become induced pluripotent stem (ips) cells. 3 reat ips cells so that they differentiate into a specific cell type.
Gene herapy Cloned gene Retrovirus capsid Bone marrow cell from patient 1 2 Insert RNA version of normal allele into retrovirus. Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. 3 Viral DNA carrying the normal allele inserts into chromosome. Bone marrow stem cells can be genetically modified in vitro to fix a genetic defect and then reintroduced back into the patient. 4 Inject engineered cells into patient. Bone marrow
Restriction Fragment Length Polymorphisms (RFLPs) Normal β-globin allele Normal allele Sickle-cell allele 175 bp 201 bp Large fragment DdeI DdeI DdeI DdeI Sickle-cell mutant β-globin allele 376 bp Large fragment DdeI DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of the β-globin gene Large fragment 201 bp 175 bp (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles 376 bp DNA from different sources cut with the same RE will result in different size DNA fragments (RFLPs)
Short andem Repeat Analysis Many parts of the human genome contain short DNA sequences repeated many times in a row (in tandem) the number of repeats in these regions is highly variable from person to person RFLP analysis can produce a DNA fingerprint that is unique for each individual (a) his photo shows Washington just before his release in 2001, after 17 years in prison. Source of sample Semen on victim Earl Washington Kenneth insley SR marker 1 17,19 16,18 17,19 SR marker 2 13,16 14,15 13,16 (b) hese and other SR data exonerated Washington and led insley to plead guilty to the murder. SR marker 3 12,12 11,12 12,12
Southern Blotting ECHNIQUE DNA + restriction enzyme Restriction fragments I II III Nitrocellulose membrane (blot) Heavy weight Gel Sponge I Normal β-globin allele 1 II Sickle-cell allele Preparation of restriction fragments III Heterozygote 2 Gel electrophoresis Alkaline solution Paper towels 3 DNA transfer (blotting) Probe base-pairs I II III with fragments I II III Radioactively labeled probe for β-globin gene 4 Nitrocellulose blot Hybridization with labeled probe Fragment from sickle-cell β-globin allele Fragment from normal β- globin allele Film over blot 5 Probe detection
Key erms for Chapter 20 recombinant DNA, gene cloning, plasmid vector transformation, ligation, restriction enzymes genomic & cdna libraries, hybridization, probe gel electrophoresis, PCR, R-PCR dideoxynucleotide, RFLP, SR, SNP in situ hybridization, DNA microarrays transgenic, stem cell, southern blot, CRISPR-Cas9 in vivo, in vitro, in situ Relevant Chapter Questions 1-10, 12