agenda 12/12/11 collect dye electrophoresis worksheet go over paper plasmid activity – do a mock...

Agenda 12/12/11• Collect dye electrophoresis worksheet• Go over paper plasmid activity – do a mock digest and

electrophoresis – look at one student’s recombinant plasmid and restriction map – pick an

enzyme to cut and talk about different sized fragments you would get• A few more slides on gene cloning/biotech• Intro 6B restriction enzyme lab – go over digest procedures (20 min)• Will check Ch. 20 notes tomorrow

Homework – Ch. 21 Notes and self-quiz due Thurs. 12/16 – use my slides to focus

on what you need to knowDNA Scissors part 2 restriction map due Wednesday – do on own, then

check key on websiteRead and do prelab 6B and be prepared to do day 1 (digest) tomorrow

– 4th will run into lunch!!!! 5th period should come in 15 minutes early, same thing for day 2

Bridging the paper plasmid activity

• 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.

• 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 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

Intro Restriction Digest Lab

Electrophoresis (aka DNA Fingerprinting)

Real World Applications • Crime scene

• Human relatedness

• Paternity

• Animal relatedness

• Anthropology studies

• Disease-causing organisms

• Food identification

• Human remains

• Monitoring transplants

Restriction enzymes• Type 2 restriction endonucleases are used in

this lab. (most common)• Cut DNA at a specific sequence.• Enzymes used

– HindIII

– EcoRI

– PstI

Why are there restriction enzymes?

• Evolved by bacteria to protect against viral infection

• Over 3000 known enzymes

Bacteriophage Lambda DNA

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

including the additional ones we will use - what were they?

– Differs from our restriction digest of the paper plasmid

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

Day 1 – Restriction Digest

Day 2 - Electrophoresis

Day 3 – Analysis of Gel

The DNA Digestion Reaction

Restriction Buffer provides optimal conditions

• NaCI provides the correct ionic strength

• Tris-HCI provides the proper pH

• Mg2+ is an enzyme co-factor

DNA Digestion Temperature

Why incubate at 37°C?

•Body temperature is optimal for these and most other enzymes

What happens if the temperature is too hot or cool?

•Too hot = enzyme may be denatured (killed)

•Too cool = enzyme activity lowered, requiring longer digestion time

Lab Procedure day 1

• Each group of 4 students sets up 4 tubes.• Handout and go through Quick Guide

procedures together• Make hypotheses about each tube

bs.kaist.ac.kr

Things to remember :

• Set up Digests on ICE!

• 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 buffer and enzymes.– DNA, then buffer, then enzymes are always last.

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

Agenda 12/13/11

• Run restriction digest – digest for 30 minutes • While waiting for digest, go over electrophoresis

procedures and prelab (last 20-30 minutes of class once all digests in water bath)

• Leave Ch. 20 notes out to be checked during period

Homework – Ch. 21 Notes and self-quiz due Thurs. 12/16 – use my

slides to focus on what you need to knowDNA Scissors worksheet due Wed – check answers with

key on website and be sure you understand!

Things to remember :• Set up Digests on ICE!

• 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 buffer and enzymes.– DNA, then buffer, then enzymes are always last.

• Reason for Enzymes being last: Enzymes are sensitive to conditions outside of their normal range. Strong buffer solutions can effect 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 none DNase activityLane 1 has a large amount of DNase activity

biosyn.com

Look at Quick Guide for Day 2

• Switch off who does pipetting for each tube – other person can be uncapping etc.

• Will do overnight staining

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

Agenda 12/14/11

• Electrophoresis of digest from yesterday and staining• I check DNA Scissors worksheet & prelab while runs• While running, you work on Analysis 1-2 and Questions

1-6,8 in lab manual

Homework – Ch. 21 Notes and self-quiz due tomorrow – use my slides

to focus on what you need to knowFinish analysis questions (except for the couple you can’t

do yet)

Lab procedure day 2

• Add loading dye to each of your 4 tubes, load marker and contents of 4 tubes and run gel.

• 1 group per gel

• Run at 100V for approx. 30 min

• Stain the gel

Agenda 12/15/11• Next 4 slides – possible errors and modified lab abstract

format• 25 min - Analyze electrophoresis lab data – put plastic wrap

on white paper and measure – Add a Table 6.3 to bottom of p. 73 for the PstI results and note the estimated base pair length of the uncut DNA in the L tube

• I check Ch. 21 while you measure, graph and fill in tables • Give actual bp data from p. 56 of BioRad manual and briefly

go over analysis questions (15 min)• Start Ch. 20 slides and 21 highlights, slides 33-71 (15 min)Homework – Finish 6B analysis to be checked tomorrowWrite abstract for lab 6 (modified format) – Transformation and

Restriction Digest – due tomorrow I suggest unless you want to work on it over break

Things that can go wrong

• Impeded digestion due to incorrect set up. (too much or too little buffer etc.)

• 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

Lab 6 Abstract

• Paragraph 1- Transformation – hypoth., results, lab error

• Paragraph 2- Digest & Electrophoresis – hypoth, results, lab error

• Paragraph 3 – Relate both to steps in Gene Cloning/Biotech Curriculum

• References• Attach – Semilog graph• Can turn in tomorrow or when back

• Comparisons among whole sets of genes and their interactions is the field of genomics.

• One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis.– Gel electrophoresis separates macromolecules -

nucleic acids or proteins - on the basis of their rate of movement through a gel in an electrical field.• Rate of movement depends on size, electrical charge,

and other physical properties of the macromolecules.

• For linear DNA molecules, separation depends mainly on size (length of fragment) with longer fragments migrating less along the gel.

Fig. 20.8

• Restriction fragment analysis indirectly detects certain differences in DNA nucleotide sequences.– After treating long DNA molecules with a restriction

enzyme, the fragments can be separated by size via gel electrophoresis.

– This produces a series of bands that are characteristic of the starting molecule and that restriction enzyme.

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

1. Restriction fragment analysis detects DNA differences that affect restriction sites

• We can use restriction fragment analysis to compare two different DNA molecules representing, for example, different alleles.– Because the two alleles must differ slightly in

DNA sequence, they may differ in one or more restriction sites.

– If they do differ in restriction sites, each will produce different-sized fragments when digested by the same restriction enzyme.

– In gel electrophoresis, the restriction fragments from the two alleles will produce different band patterns, allowing us to distinguish the two alleles.

• Restriction fragment analysis is sensitive enough to distinguish between two alleles of a gene that differ by only 1 base pair in a restriction site.

Fig. 20.9

HERE WE ARE RUNNING PURIFIED SEGMENT OF DNA WITH GENE OF INTEREST

• 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.

• 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.

• For our three individuals, the results of these steps show that individual III has a different restriction pattern than individuals I or II. Here we are running all DNA but probe identifies the fragments that have to do with our gene of interest.

Fig. 20.10

• Southern blotting can be used to examine differences in noncoding DNA as well.

• Differences in DNA sequence on homologous chromosomes that produce different restriction fragment patterns are scattered abundantly throughout genomes, including the human genome.

• These restriction fragment length polymorphisms (RFLPs) can serve as a genetic marker for a particular location (locus) in the genome.– A given RFLP marker frequently occurs in

numerous variants in a population.

• RFLPs are detected and analyzed by Southern blotting, frequently using the entire genome as the DNA starting material.– These techniques will detect RFLPs in

noncoding or coding DNA.

• Because RFLP markers are inherited in a Mendelian fashion, they can serve as genetic markers for making linkage maps.– The frequency with which two RFPL markers -

or a RFLP marker and a certain allele for a gene - are inherited together is a measure of the closeness of the two loci on a chromosome.

DNA MICROASSAYS

• Automation has allowed scientists to detect and measure the expression of thousands of genes at one time using DNA microarray assays.– Tiny amounts of a large number of single-

stranded DNA fragments representing different genes are fixed on a glass slide in a tightly spaced array (grid).

– The fragments are tested for hybridization with various samples of fluorescently-labeled cDNA molecules.

Fig. 20.14a

• Spots where any of the cDNA hybridizes fluoresce with an intensity indicating the relative amount of the mRNA that was in the tissue.

Fig. 20.14b

• Ultimately, information from microarray assays should provide us a grander view: how ensembles of genes interact to form a living organism.– It already has confirmed the relationship

between expression of genes for photosynthetic enzymes and tissue function in leaves versus roots of the plant Arabidopsis.

– In other cases, DNA microarray assays are being used to compare cancerous versus noncancerous tissues.• This may lead to new diagnostic techniques and

biochemically targeted treatments, as well as a fuller understanding of cancer.

• Perhaps the most interesting genes discovered in genome sequencing and expression studies are those whose function is completely mysterious.

• One way to determine their function is to disable the gene and hope that the consequences provide clues to the gene’s normal function.– Using in vitro mutagenesis, specific changes

are introduced into a cloned gene, altering or destroying its function.

– When the mutated gene is returned to the cell, it may be possible to determine the function of the normal gene by examining the phenotype of the mutant.

• In nonmammalian organisms, a simpler and faster method, RNA interference (RNAi), has been applied to silence the expression of selected genes.– This method uses synthetic double-stranded

RNA molecules matching the sequences of a particular gene to trigger breakdown of the gene’s mRNA.

– The mechanism underlying RNAi is still unknown.

– Scientists have only recently achieved some success in using the method to silence genes in mammalian cells.

• The next step after mapping and sequencing genomes is proteomics, the systematic study of full protein sets (proteomes) encoded by genomes.– Difficult due to vast number

• Genomic and proteomics are giving biologists an increasingly global perspective on the study of life.

• Advances in bioinformatics, the application of computer science and mathematics to genetic and other biological information, will play a crucial role in dealing with the enormous mass of data.

• These analyses will provide understanding of the spectrum of genetic variation in humans.– Because we are all probably descended from a small

population living in Africa 150,000 to 200,000 years ago, the amount of DNA variation in humans is small.

– Most of our diversity is in the form of single nucleotide polymorphisms (SNPs), single base-pair variations.• In humans, SNPs occur about once in 1,000 bases, meaning

that any two humans are 99.9% identical.

– The locations of the human SNP sites will provide useful markers for studying human evolution and for identifying disease genes and genes that influence our susceptibility to diseases, toxins or drugs.

Ch. 21 Highlights – Genes and Development

Differential gene expression leads to different cell types in multicellular organisms

• Zygote undergoes transformation through 3 interrelated cell processes

1) Cell division – mitosis increases # of cells

2) Cell differentiation – cells become specialized in structure and function

3) Morphogenesis – organization of cells into tissues and organs

What are the terms that describe an embryo going through these stages? (Remember Ch. 47)

Fig. 21.2

– cell lineage, a type of fate map.• A fate map traces the development of an embryo.

CHAPTER 21 THE GENETIC BASIS OF

DEVELOPMENT

Section B: Differential Gene Expression

1. Different types of cells in an organism have the same DNA

2. Different cell types make different proteins, usually as a result of

transcriptional regulation

3. Transcriptional regulation is directed by maternal molecules in the

cytoplasm and signals from other cells

• The differences between cells in a multicellular organism come almost entirely from differences in gene expression, not differences in the cell’s genomes.

• These differences arise during development, as regulatory mechanisms turn specific genes off and on.

• An important question that emerges is whether genes are irreversibly inactivated during differentiation.

Differing Gene Expression

• Much evidence supports the conclusion that nearly all the cells of an organism have genomic equivalence - that is, they all have the same genes.

• An important question that emerges is whether genes are irreversibly inactivated during differentiation.

1. Different types of cell in an organism have the same DNA

In many plants, whole new organisms can develop from differentiated somatic cells.During the 1950s, F.C. Steward and his students found that differentiated root cells removed from the root could grow into normal adult plants when placed in a medium culture.

•These cloning experiments produced genetically identical individuals, popularly called clones.

In plants, at least, cell can remain totipotent.

•Plant cloning is now used extensively in agriculture.

Animal Cloning Much Harder• In tadpoles, the ability of

the transplanted nucleus to support normal development is inversely related to the donor’s age.

• 1997 when Ian Wilmut and his colleagues cloned an adult sheep

• One, “Dolly,” of several hundred implanted embryos completed normal development. – Improper

methylation in many cloned embryos interferes with normal development.

• Stem cells that can differentiate into multiple cell types are multipotent or, more often, pluripotent.

• Embryonic vs. adult– embryonic stem cells

are “immortal” because of the presence of telomerase that allows these cells to divide indefinitely.

• Under the right conditions, cultured stem cells derived from either source can differentiate into specialized cells.

Stem Cells

• Beyond the study of differentiation, stem cell research has enormous potential in medicine.

• The ultimate aim is to supply cells for the repair of damaged or diseased organs.– For example, providing insulin-producing pancreatic

cells to diabetics or certain brain cells to individuals with Parkinson’s disease could cure these diseases.

• At present, embryonic cells are more promising than adult cells for these applications.

• However, because embryonic cells are derived from human embryos, their use raises ethical and political issues.

More Ch. 21• What controls differentiation

and morphogenesis?1) Cytoplasmic determinants – maternal substances in egg that are distributed unevenly in early cells of embryo

2) Cell-cell signals result from molecules, such as growth factors, that are produced by one cell to influence neighboring cells – called induction. – Causes cells to differentiate

Fig. 21.9

Determination – series of events that lead to observable differentiation of a cell. Differentiation is caused by cell-cell signals and is irreversible. When cell is irreversible committed to its final fate, it is said to be “determined.”

More Ch. 21Pattern formation – sets up the body plan as a

result of cytoplasmic determinants and inductive signals.– Determines head/tail, left/right, back/front– Uneven distribution of substances called

morphogens plays a role in establishing these axes.

• Bicoid mRNA- a morphogen– Concentrated at extreme

anterior end

More Ch. 21

• Homeotic genes – master regulatory genes that determine the location and organization of body parts, esp. appendages of each segmentHox genes = one class of Homeotic genes (include 180

nucleotide sequence called homeobox)– Highly conserved in evolution

- changes in these genes and the genes that regulate them can have a profound effect on morphology, leading to evolutionary change

ex: variable expression of a Hox gene in a fish fin bud and a chicken leg bud leads to longer skeletal extension in chicken leg compared to fish fin

• In fact, the vertebrate genes homologous to the homeotic genes of fruit flies have even kept their chromosomal arrangement.

Fig. 21.15

Apoptosis (Cell suicide) and Normal Development

• A built-in cell suicide mechanism is essential to development in all animals.– Similarities between the apoptosis genes in

mammals and nematodes indicate that the basic mechanism evolved early in animal evolution.

– The timely activation of apoptosis proteins in some cells functions during normal development and growth in both embryos and adults.• It is part of the normal development of the

• Problems with the cell suicide mechanism may have health consequences, ranging from minor to serious.– Failure of normal cell death during

morphogenesis of the hands and feet can result in webbed fingers and toes.

– Researchers are also investigating the possibility that certain degenerative diseases of the nervous system result from inappropriate activation of the apoptosis genes.

– Others are investigating the possibility that some cancers result from a failure of cell suicide which normally occurs if the cell has suffered irreparable damage, especially DNA damage.

Agenda 12/16/11• Finish slides from yesterday (if we don’t finish, you need to finish on

your own over break and write down any questions you have for first day back)

• Collect lab reports that are done• Check that 6B Analysis is done in lab manual

Homework – Write abstract for lab 6 – Transformation and Restriction Digest - if not

done Review Manual - Molecular Genetics (Ch. 11) and Lab 6 & 7 due Tues

1/3Molecular Genetics/Biotech Test Thurs. 1/5Will also cover Ch. 22-25 before finals so could get started on those.

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