Agenda 1/14 Restriction digests – next 2 slides then put up slide 4 of DNA Fingerprinting while...

Agenda 1/14• Restriction digests – next 2 slides then put

up slide 4 of DNA Fingerprinting while working – answer questions on page 30 of packet as you work!!!

Once all samples incubating:• Procedure for tomorrow (Biorad slides 14-18) & then back to these

slides

Homework – Notes and concept checks for 21.2 and 21.5 due Wednesday, online

due Friday)Read and highlight quickguide procedure for lab tomorrow -

be ready to do electrophoresis tomorrow!Cell Respiration and Photosynthesis Review Manual due tomorrow,

Molecular Biology due Tuesday 1/22

Things to remember :• Set up Digests on ICE!• Take turns pipetting and everyone helps!!!• Concentrations are important (More is not better)

– THESE ARE TINY AMOUNTS – BE SURE YOUR TIP IS IN BOTTOM AND YOU GET IT ALL – NO AIR BUBBLES!!!

• Add reagents in Correct order and use fresh tip for each sample and enzymes.– DNA, then enzymes are always last.

• Reason for Enzymes being last: Enzymes are sensitive to conditions outside of their normal range. Strong buffer solutions can affect the efficiency of the digest

• Keep it clean and don’t digest too long– Contaminations of DNases can be

devastating.

Lane 8 has little to no DNase activityLane 1 has a large amount of DNase activity

biosyn.com

Concept 20.2: DNA technology allows us to study the sequence,

expression, and function of a gene• DNA cloning allows researchers to

– Compare genes and alleles between individuals

– Locate gene expression in a body

– Determine the role of a gene in an organism

• Several techniques are used to analyze the DNA of genes

© 2011 Pearson Education, Inc.

Gel Electrophoresis and Southern Blotting

• One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis

• This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size, electrical charge, and other properties

• A current is applied that causes charged molecules to move through the gel

• Molecules are sorted into “bands” by their size


Figure 20.9a

Mixture ofDNA mol-ecules ofdifferentsizes

Powersource

Powersource

Longermolecules

Cathode Anode

Wells

Gel

Shortermolecules

TECHNIQUE

2

1

Figure 20.9b

RESULTS

–The separated fragments can be recovered undamaged from gels, providing pure samples of individual fragments.

• Variations in DNA sequence are called polymorphisms

• Sequence changes that alter restriction sites are called RFLPs (restriction fragment length polymorphisms)


Figure 20.10

Normal -globin allele

Sickle-cell mutant -globin allele

Largefragment

Normalallele

Sickle-cellallele

201 bp175 bp

376 bp

(a) DdeI restriction sites in normal andsickle-cell alleles of the -globin gene

(b) Electrophoresis of restrictionfragments from normal andsickle-cell alleles

201 bp175 bp

376 bp

Large fragment

Large fragment

DdeI DdeI DdeI DdeI

DdeI DdeI DdeI

NOTE - HERE WE ARE RUNNING PURIFIED SEGMENT OF DNA WITH GENE OF INTEREST, SIMPLE LIKE OUR LAB

• Gel electrophoresis combined with nucleic acid hybridization allows analyses to be conducted on the whole genome, not just cloned and purified genes.

• Although electrophoresis will yield too many bands to distinguish individually, we can use nucleic acid hybridization with a specific probe to label discrete bands that derive from our gene of interest.

• The radioactive label on the single-stranded probe can be detected by autoradiography, identifying the fragments that we are interested in.

Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

• A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization

• Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA of our gene of interest, immobilized on a “blot” of gel


Figure 20.11

DNA restriction enzyme

321

4

TECHNIQUE

I Normal-globinallele

II Sickle-cellallele

III Heterozygote

Restrictionfragments

Nitrocellulosemembrane (blot)

Heavyweight

Gel

Sponge

Alkalinesolution Paper

towels

III III

III III III III

Preparation ofrestriction fragments

Gel electrophoresis DNA transfer (blotting)

Radioactively labeledprobe for -globingene

Nitrocellulose blot

Probe base-pairswith fragments

Fragment from sickle-cell -globin allele

Fragment from normal - globin allele

Filmoverblot

Hybridization with labeled probe Probe detection5

• We can tie together several molecular techniques to compare DNA samples from three individuals.– We start by adding the restriction enzyme to each of

the three samples to produce restriction fragments.– We then separate the fragments by gel

electrophoresis.– Southern blotting (Southern hybridization) allows us

to transfer the DNA fragments from the gel to a sheet of nitrocellulose paper, still separated by size.• This also denatures the DNA fragments.

– Bathing this sheet in a solution containing our probe allows the probe to attach by base-pairing (hybridize) to the DNA sequence of interest and we can visualize bands containing the label with autoradiography.


Verbal description of previous picture

How do we get enough DNA to work with from small samples?

• PCR

• http://www.dnalc.org/resources/spotlight/index.html (scroll down and watch both 2D and 3D animations)

http://www.dnalc.org/resources/spotlight/index.html



• DNA cloning is the best method for preparing large quantities of a particular gene or other DNA sequence.

• When the source of DNA is scanty or impure, the polymerase chain reaction (PCR) is quicker and more selective.

• This technique can quickly amplify any piece of DNA without using cells.

The polymerase chain reaction (PCR) clones DNA entirely in vitro


• The DNA is incubated in atest tube with special DNA polymerase, a supply of nucleotides,and short pieces ofsingle-stranded DNA as a primer.


Fig. 20.7

• PCR can make billions of copies of a targeted DNA segment in a few hours.– This is faster than cloning via recombinant bacteria.

• In PCR, a three-step cycle: heating, cooling, and replication, brings about a chain reaction that produces an exponentially growing population of DNA molecules.– The key to easy PCR automation was the discovery of

an unusual DNA polymerase, isolated from bacteria living in hot springs, which can withstand the heat (Taq polymerase) needed to separate the DNA strands at the start of each cycle.


• Devised in 1985, PCR has had a major impact on biological research and technology.

• PCR has amplified DNA from a variety of sources:– fragments of ancient DNA from a 40,000-year-

old frozen wooly mammoth,– DNA from tiny amount of blood or semen found

at the scenes of violent crimes,– DNA from single embryonic cells for rapid

prenatal diagnosis of genetic disorders,– DNA of viral genes from cells infected with

difficult-to-detect viruses such as HIV. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

1/15• Electrophoresis – next 2 slides then put up DNA

Fingerprinting slide 5 while working• Once all gels running:

– Collect review manual answers– Gene cloning

Homework – Lab packet pgs. 34,35,40Notes and concept checks for 21.2 and 21.5 due tomorrow,

NO ONLINE ASSIGNMENT UNTIL NEXT SEMESTERMolecular Biology Review Manual due Tuesday 1/22You should be studying, doing test corrections, etc.

Lab procedure day 2

• Remember to change dials on micropipettes as needed for different amounts- follow directions carefully!

• Add loading dye to each of your tubes, load marker and contents of 6 tubes and run gel.

• 1 group per gel• Run at 100V for approx. 30 min • Stain the gel

How to load wells

Biotech Vocab.

• genetic engineering - the direct manipulation of genes for practical purposes.

• recombinant DNA - genes from two different sources - often different species - are combined in vitro into the same molecule

• biotechnology - the manipulation of organisms or their components to make useful products

• gene cloning – preparing multiple identical copies of gene-sized pieces of DNA

• cloning vector - DNA molecule that can carry foreign DNA into a cell and replicate there

Bacterium

Bacterialchromosome

Plasmid

2

1

3

4

Gene inserted intoplasmid

Cell containing geneof interest

RecombinantDNA (plasmid)

Gene of interest

Plasmid put intobacterial cell

DNA ofchromosome(“foreign” DNA)

Recombinantbacterium

Host cell grown in culture toform a clone of cells containingthe “cloned” gene of interest

Gene of interest

Protein expressed fromgene of interest

Protein harvestedCopies of gene

Basic researchand variousapplications

Basicresearchon protein

Basic research on gene

Gene for pestresistance insertedinto plants

Gene used to alterbacteria for cleaningup toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growthhormone treatsstunted growth

DNA Technology- Gene Cloning

• The potential uses of cloned genes fall into two general categories.

• First, the goal may be to produce a protein product.– For example, bacteria carrying the gene for human

growth hormone can produce large quantities of the hormone for treating stunted growth.

• Alternatively, the goal may be to prepare many copies of the gene itself.– This may enable scientists to determine the gene’s

nucleotide sequence or provide an organism with a new metabolic capability by transferring a gene from another organism.


• The process of cloning a human gene in a bacterial plasmid can be divided into five steps.


Fig. 20.3

http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html

Good narration/animation of this process



1. Isolation of vector and gene-source DNA.

• The source DNA comes from human tissue cells.

• The source of the plasmid is typically E. coli.– This plasmid carries two useful genes, ampR,

conferring resistance to the antibiotic ampicillin and lacZ, encoding the enzyme beta-galactosidase which catalyzes the hydrolysis of sugar.

– The plasmid has a single recognition sequence, within the lacZ gene, for the restriction enzyme used.Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

2. Insertion of DNA into the vector.

• By digesting both the plasmid and human DNA with the same restriction enzyme we can create thousands of human DNA fragments, one fragment with the gene that we want, and with compatible sticky ends on bacterial plasmids.

• After mixing, the human fragments and cut plasmids form complementary pairs that are then joined by DNA ligase.

• This creates a mixture of recombinant DNA molecules.


Figure 20.3-3

Recombinant DNA molecule

One possible combinationDNA ligaseseals strands

DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.

Restriction enzymecuts sugar-phosphatebackbones.

Restriction site

DNA5

5

5

5

5

5

5

5

55

5

5

55

5

5

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

2

3

1

Sticky end

GAATTCCTTAAG

CTTAAG AATTC

G

GGAATTC

CTTAA

GG

GG

AATT CAATT CC TTAA C TTAA

3. Introduction of the cloning vector into cells.

• Bacterial cells take up the recombinant plasmids by transformation.– These bacteria are lacZ-, unable to hydrolyze

lactose.

• This creates a diverse pool of bacteria, some bacteria that have taken up the desired recombinant plasmid DNA, other bacteria that have taken up other DNA, both recombinant and nonrecombinant.


4. Cloning of cells (and foreign genes).

• We can plate out the transformed bacteria on solid nutrient medium containing ampicillin and a sugar called X-gal.– Only bacteria that have the ampicillin-

resistance plasmid will grow.– The X-gal in the medium is used to identify

plasmids that carry foreign DNA.• Bacteria with plasmids lacking foreign DNA stain

blue when beta-galactosidase hydrolyzes X-gal.• Bacteria with plasmids containing foreign DNA are

white because they lack beta-galactosidase.


5. Identifying cell clones with the right gene.

• In the final step, we will sort through the thousands of bacterial colonies with foreign DNA to find those containing our gene of interest.

• One technique, nucleic acid hybridization, depends on base pairing between our gene and a complementary sequence, a nucleic acid probe, on another nucleic acid molecule.– The sequence of our RNA or DNA probe depends on knowledge

of at least part of the sequence of our gene.– A radioactive or fluorescent tag labels the probe.

• The probe will hydrogen-bond specifically to complementary single strands of the desired gene.– After denaturation

(separating) the DNA strands in the plasmid, the probe will bind with its complementary sequence, tagging colonies with the targeted gene.

Fig. 20.4

• What if we put whole eukaryotic gene into a prokaryote?– What can’t they do? (see next slide)

Fig. 20.5

Continuing on gene cloning…

• Complementary DNA(cDNA) is DNA made in vitro using mRNA as a template and the enzyme reverse transcriptase. -impt. since bacteria can’t do mRNA processing

• A complete set of recombinant plasmid clones, each carrying copies of a particular segment from the initial genome, forms a genomic library.– The library can be saved and used as a source

of other genes or for gene mapping.

• cDNA libraries also exist– Limitations are every cell only expressed certain

mRNA’s at a time, so won’t get every gene

Cloned genes are stored in DNA libraries

• In addition to plasmids, certain bacteriophages are also common cloning vectors for making libraries.– The recombinant phage

DNA is packaged in a capsid in vitro and allowed to infect a bacterial cell.

– Infected bacteria produce new phage particles, each with the foreign DNA.


1/16• Cells Warm up question, slide 15• Review packet 34,35,40• Get gel back then do next slides to explain stepwise• Interpret results, use packet pgs. 41-46• I check Ch. 21 notes while you work• DNA Fingerprinting slide 19

Homework:Finish DNA Fingerprinting lab packetRead and highlight plasmid mapping extension activity pgs. 47-49 –

you will finish this with your group tomorrow Molecular Biology Review Manual due Tuesday 1/22You should be studying, doing test corrections, etc.

Me – have boxes with rulers, sharpies, white paper for background and have one student from each group glove and drain gel, then gel in ziploc for measuring. (can use transparency film to trace if time)

get plasmid model from Ms. Kaehms

Bacteriophage Lambda DNA

• Show picture p. 31 of BioRad Instruction Manual– Shows where HindIII cuts – Similar maps exist for other restriction enzymes,

including the ones we used - what were they?– Differs from our restriction digest of the

plasmids• If plasmid cut once, how many fragments?• If linear phage DNA cut once, how many fragments?

Making a standard curve

• Measure to leading edge of each band in the marker/ ladderQuickTime™ and a

decompressorare needed to see this picture.

Standard Curve

• Graph of lambda HindIII marker (base pair vs. Distance migrated)

• Using semi log graph paper

QuickTime™ and a decompressor

are needed to see this picture.

Using graph to find size of Unknowns



Things that can go wrong

• Impeded digestion due to incorrect set up

• Star digest activity– Under non-standard reaction

conditions, some restriction enzymes are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. This altered specificity has been termed “star activity"

Star digestion example

• Examples:

– EcoRI is supposed to only cut GAATTC but, under extreme conditions, it might possibly cut CAATTC also.



http://www.fermentas.com/en/support/technical-reference/restriction-enzymes/star-activity

Star digests

• Things that can cause Star digests– Too much glycerol in reaction. (More is not

better)– Incorrect buffer or buffer concentration– Extended digest times. Don’t leave them over

night

1/17

• Plasmid mapping extension using packet• 10 min until end of period, go over 1-6 (do print screen of

S2 plasmid and draw to explain answers)

Homework – Finish both lab packets as needed and be ready to wrap up

tomorrow, updated Powerpoints all online by end of today

Molecular Biology review manual due Tuesday 1/22

1/18

• Wrap up lab packets• Relatedness DNA fingerprints – parent/child• Finish gene cloning (back to slide 33) and viruses

Homework – In exchange for no lab write-up/quiz, you must go over

weekly Powerpoints and Big Idea Powerpoint for Molecular Bio, then look back at old Big Idea Powerpoints --- STUDY!!!

Molecular Genetics Review Manual due Tuesday

Expected results

Determining Gene Function• One way to determine function is to disable the

gene and observe the consequences• Using in vitro mutagenesis, mutations are

introduced into a cloned gene, altering or destroying its function

• When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype


• Gene expression can also be silenced using RNA interference (RNAi)

• Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the gene’s mRNA

Purple petunias• http://www.pbs.org/wgbh/nova/body/rnai.html

14 min (start at 2:45 if less time)


http://www.pbs.org/wgbh/nova/body/rnai.html

• In humans, researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition

• Genetic markers called SNPs (single nucleotide polymorphisms) occur on average every 100–300 base pairs

• In humans, SNPs occur about once in 1,000 bases, meaning that any two humans are 99.9% identical.

• SNPs can be detected by PCR, and any SNP shared by people affected with a disorder but not among unaffected people may pinpoint the location of the disease-causing gene


Figure 20.16

DNA

SNPNormal allele

Disease-causingallele

T

C

• Genomics is the study of whole sets of genes and their interactions

• Proteomics is the systematic study of all proteins encoded by a genome

• Bioinformatics is the application of computational methods to the storage and analysis of biological data


Viruses

• Evolved from plasmids or transposons most likely (mobile genetic material) – more closely related to host cell DNA than other virus DNA

• Probably entered damaged cells first, then once developed capsid/envelope, could enter undamaged cells also

• NOTE- AP CURRICULUM FOCUSES ON HOW THEY CAN INTRODUCE GENETIC VARIATION

Viruses - Relative size and structure

Viral Genome Possibilities

• The capsid is a protein shell enclosing the viral genome.

• Capsids are build of a large number of protein subunits called capsomeres, but with limited diversity.– The capsid of the tobacco

mosaic virus has over 1,000 copies of the same protein.

– Adenoviruses have 252 identical proteins arranged into a polyhedral capsid - as an icosahedron.

Fig. 18.2a & b

• Some viruses have viral envelopes, membranes cloaking their capsids.

• These envelopes are derived from the membrane of the host cell.

• They also have some viral proteins and glycoproteins.

Fig. 18.2c

• The most complex capsids are found in viruses that infect bacteria, called bacteriophages or phages.

• The T-even phages that infect Escherichia coli have a 20-sided capsid head that encloses their DNA and protein tail piece that attaches the phage to the host and injects the phage DNA inside.

Fig. 18.2d

• Can reproduce only within a host cell.• An isolated virus is unable to reproduce - or do

anything else, except infect an appropriate host.• Host range - “lock-and-key” fit between proteins on

the outside of virus and specific receptor molecules on the host cell’s surface.

• Some viruses (like the rabies virus) have a broad enough host range to infect several species, while others infect only a single species.

• Most viruses of eukaryotes attack specific tissues.

Viruses- Living or Not?

• Virus takes over the machinery of the host cell!

Fig. 18.3

Virulent Phages and the Lytic Cycle

• While phages have the potential to wipe out a bacterial colony in just hours, bacteria have defenses against phages.– Natural selection favors bacterial mutants with

receptors sites that are no longer recognized by a particular type of phage.

– Bacteria produce restriction nucleases that recognize and cut up foreign DNA, including certain phage DNA.• Modifications to the bacteria’s own DNA prevent its

destruction by restriction nucleases.

– But, natural selection favors resistant phage mutants.

Temperate Phages Use Lytic and Lysogenic Cycle

• Human immunodeficiency virus (HIV)

• Causes AIDS (acquired immunodeficiency syndrome)

• Includes an envelope with glyco-proteins for binding to specific types of white blood cells

Fig. 18.7a

Retroviruses

• After HIV enters the host cell, reverse transcriptase synthesizes double stranded DNA from the viral RNA.

• Transcription produces more copies of the viral RNA that are translated into viral proteins, which self-assemble into a virus particle and leave the host.

Fig. 18.7b

• Vaccines can help prevent viral infections– Edward Jenner and 1st vax for smallpox late

1700’s – used cowpox from milkmaid’s sore

• Antibiotics only work on bacteria, not viruses

• Antivirals interfere with viral nucleic acid synthesis– AZT interferes with reverse transcriptase of HIV.– Acyclovir inhibits herpes virus DNA synthesis.

3 Causes:1) Mutation of existing viruses

– RNA viruses tend to have high mutation rates because replication of their nucleic acid lacks proofreading.

– Some mutations create new viral strains with sufficient genetic differences from earlier strains • This is the case in flu epidemics.

2) Spread of existing viruses from one host species to anotherex. – Hantavirus from mice to people

3) Spread from a small, isolated population to a widespread epidemic.

ex. - AIDS

Emergence of New Viral Diseases

• Viral diseases in a small isolated population can emerge and become global

• New viral diseases can emerge when viruses spread from animals to humans

• Viral strains that jump species can exchange genetic information with other viruses to which humans have no immunity

• These strains can cause pandemics, global epidemics

• The 2009 flu pandemic was likely passed to humans from pigs; for this reason it was originally called the “swine flu”


Agenda 1/22/13• Folders back • Big Idea Powerpoint practice questions slides 33-37 - I check review manual

while you work• For extra review: http://www.dnai.org/a/index.html - go to Code, then for

each of the 4 tabs at the bottom, go to putting the pieces together- great animations and practice modules

Homework – Have last 2 lab packets (CSI and plasmid mapping) ready to hand in on final

dayStudy for Final – blue/black pen, #2 pencils, and binder paper (3 sheets

stapled) requiredMath = rate, biomass, lag time, Chi square, fragment lengthsFRQ’s = Protein synthesis/mutation/genetic disease, Gene regulation, Recombinant DNA technologyMC = 2/3 Molecular Genetics, 1/3 cumulative

http://www.dnai.org/a/index.html

Final Day• Collect lab packets (5 min)• MC (65 min)• 10 min break• FRQ (5 min reading period, then 34 min writing)• If time:• Funny:

http://www.youtube.com/watch?v=CQEaX3MiDow - GTCA

http://www.youtube.com/watch?v=x5yPkxCLads&NR=1 - a tribute to PCR

http://www.youtube.com/watch?v=dIZpb93NYlw&feature=related - 2 students

http://www.youtube.com/watch?v=CQEaX3MiDow



http://www.youtube.com/watch?v=x5yPkxCLads&NR=1



http://www.youtube.com/watch?v=dIZpb93NYlw&feature=related



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