Module 3 Exam Review

Ribosomal RNA genes

-regions of high conservation and regions of high variability -useful for phylogenetic and evolutionary studies -correlate ribosomal sequence types (rRNA genotypes) with variable pathogenicity

Cloned sequence characterization

-restriction mapping -nucleic acid blotting -DNA sequencing

Northern blotting

-screen mRNA to determine expression characteristics of a gene -the probe is single stranded DNA derived from a cloned copy of the gene of interest

Arabidopsis genome

-small genome -many gene duplications -used as a model organism -higher plants have larger genomes but approx. the same amount of genes

restriction mapping

-use RE to determine location, distance, and order of RE sites in a clone -use single and double digests -construct map of RE locations based on results

Microsatellite analysis

-using 8 dog micro satellites -use SNPs for variable markers -those are in front of varying numbers of repeats (micro satellites) - the microsatellite marker used can now discriminate between all three males due to the variability of the number of repeats

A linear DNA fragment is cut with a restriction enzyme to yield two fragments. There is/are __ site(s) for this enzyme in this fragment.

1

YAC vector process explained

Cleave YAC. Has left and right arm that has a telomere on the end. Insert DNA > 100 kB

It is common to use ddNTPs (dideoxyribonucleoside triphosphates) in which of the following biochemical reactions?

DNA sequencing

Taq polymerase

- key to PCR, derived from thermophilic bacteria Thermus aquaticus - hot spring bacteria, the polymerase works at elevated temps: 69-72C - Taq polymerase survives at high temps (such as 95C)

There are different challenges that exist for sequencing prokaryotic and eukaryotic genomes. Which challenge is correctly paired with the type of genome to which it relates?

Eukaryotic: repetitive DNA

A ddNTP, used often in DNA sequencing, lacks a(n) ________ at the ________ and ________ carbons.

OH, 2', 3'

In the context of molecular genetics, reverse translation refers to

assembling a DNA sequence from an amino acid sequence.

Nucleic acid blotting is widely used in recombinant DNA technology. In a Southern blot one generally

hybridizes filter-bound DNA with a DNA probe.

Fragment ligation

join the ends of DNA together - complimentary ("sticky") ends will anneal with one another - 2 different molecules cut with the same RE can anneal to one another -annealing allows recombinant DNA molecules to form by complementary base pairing. The two strands are not covalently bonded. -DNA ligase seals those gaps which covalently bond the two strands

microarray analysis

label mRNAs and extract samples from disease and label with another color

ß-galactosidase

lacZ

Compared with prokaryotic chromosomes, eukaryotic chromosomes are

large, linear, less densely packed with protein-coding genes, mainly organized in monocistronic units with introns.

Typically, bacterial DNA contains_____ (more or less?) repetitive DNA than eukaryotic DNA.

less

cDNA libraries

library of genes active in particular cell type or a particular time

What is meant by the term low gene density? Give an example of an organism with low gene density.

low ratio of number of genes per base pairs; humans

When propagating a clone in the lambda phage, would you have more immediate success if the phage entered the lysogenic or the lytic cycle?

lytic because it bursts cells and makes more phages

E. coli

prokaryotic model organism

Which of the following enzymes is used to make complementary DNA (cDNA) from RNA?

reverse transcriptase

Ionome

study of ions in organisms, eg., Fe, Mn, Mg, K, Cu, Ca, Ni, S etc.

In situ hybridization of mRNA

see Hoxc11 mRNA distribution in development - protein product of that gene is a transcription factor critical for normal early development

hematopoietic stem cells

stem cells in the bone marrow that can give rise to a large number of different blood cell types. However, there lineages are restricted to blood cells.

DNA sequencing

ultimate characterization of a clone - determine complete primary sequence of the clone

dog cloning results

- 2 live births - 1 dog survived (snippy) - inefficient 1/1095 success rate or 2 dogs from 123 recipients

early results

- 4 months post-infusion: sick cell patient had no vaso-occlusive events ( had 7 in previous year), total hemoglobin level rose - 9 months post infusion: beta thalassemia patient, no transfusions, fetal globin was about 47% of total - promising early results, more study needed - side effects were result of bisulfan chemotherapy

leptin knockout example

- A functional leptin protein and a functional leptin receptor are both needed for normal metabolism - Boy had homozygous base deletion in the leptin gene. Mutation results in a frameshift mutation and does not produce functional leptin which results in serious metabolic issues - used recombinant human leptin on boy. Normal metabolic function of the pathway was restored and disorder reversed. - Through gene knockout techniques, we can create mice that lack the leptin receptor (db-)

Plasmid cloning overview

- A plasmid vector is removed from bacterial cell and cut with a restriction enzyme - DNA to be cloned is cut with the same restriction enzyme - The two DNAs are ligated to form a recombinant molecule - The plasmid with the clone DNA is introduced into host cell - cells are selected for carrying recombinant plasmids by plating on medium with antibiotic or color indicator

Artificial chromosomes

- Bacterial artificial chromosomes (BACs) - good for large fragments - based on F(ertility) factor of bacteria -clone up to 300 kb (great) -multiple cloning sites, selective markers, promoters to express inserted genes

Library screening

- Clones from library grown on nutrient plate - Replica plate made by transfer to filter - Transferred bacteria lysed, immobilized on filter, and DNA made single stranded - Colony DNA then screened with DNA probe (labeled in some way: radioactive, fluorescent) - Probe can be similar gene from another organism or synthetically derived from partial amino acid sequence from a protein of interest - Excess probe washed off filter - If radioactive probe used, place film on filter - Compare position of positive colony to original plate to determine colony of interest - Pick original colony, transfer to growth medium, analyze further (you grow bacteria from a single positive colony)

Advancements in Knockout Technology

- Cre-Lox system for conditional tissue-specific knockout to targeted gene - One line of mice contains conditional allele of gene ("floxed") - another line contains a Cre recombinase gene under control of TISSUE SPECIFIC PROMOTER -cross lines, induce Cre, targeted gene "knocked out" in that tissue

methods of isolating nucleic acids

- DNA isolated from cells - many methods to isolate nucleic acids - fractionation methods that separate nucleic acids from proteins and other cellular debris -many simple commercial kits (Qiagen) available for this today to extract DNA from countless sources

BCL11A

- Down regulates expression of fetal globin after birth - mutants of BCL11A found that result in adult expression of fetal hemoglobin - BCL11A target of CRISPR Cas in current clinical trials on sickle cell patients and Beta thalassemia patients

Drosophila as a model organism

- Drosophila advantages - ease of growth - size of its genome - short generation time (about ten days) - large fecundity (about 3000 offspring in a lifetime) - moderate crossing over in females - no crossing over in males ----- these simplify genetic analysis

working with stem cells

- ES cells are easier to grow and are totipotent - Adult stem cells are less well characterized- their full potential is not yet known (but they could provide a non-controversial solution to avoid the ethical issues that arise when human embryos are involved)

Flu strain designations

- Flu strains placed into different groups based on two types of surface proteins HA and NA - 16 types of HA and 9 types of NA - any combo of HA and NA can infect birds - H1, H2, H3, H5, H7, H9 and the N1, N2, and N7 surface proteins were known to infect humans

controversy with ES cells

- For most of the 2000s federally funded research using Human ES cells was restricted to a handful of previously generated ES lines - Work on newly derived ES cells had to be funded through private or state mechanisms - recently, a series of landmark papers described how keratinocytes, or skin cells, could be made into ES cells - this will allow a great deal of scientific experiments on their differentiation and their potential for therapeutic use

Human Chimpanzee chromosome comparison

- Human 21 and chimp 22 share 179 genes with coding sequences of identical length - these shared genes are 99.29% similar at nucleotide level, 99.18% at amino acid level

Other uses of PCR

- Identification of RE variants - Microsatellite analysis - Screening for genetic disorders - Diagnostic screening for infectious organisms (PCR reaction disseminated an infection in a dog (Booton)) ----can also screen for influenza - Forensics - Paleobiology

LacZ insert identification

- LacZ gene is part of bacterial Lac operon - Produces Beta-galatosidase that metabolizes lactose to produce glucose and galactose - Beta-galactosidase action results in a blue by-product when X-gal is present in the growth medium (color indicator)

Generation of ES stem cell lines

- Mate mice - isolate blastocysts - plate with feeder cells and LIF containing media, maintains undifferentiated state - isolate and passage cells of inner cell mass - continue passage of ES lines - ES cells can be reinfected into mouse embryos and will populate all the tissues. An embryo can be created solely from ES cells.

PCR

- Polymerase Chain Reaction -Cloning without need for host cell - making many copies (millions)

Candidate genes (reverse genetics)

- Potential candidate genes may be subjected to further genetic and biochemical analysis, such as: - DNA database searching and sequence alignment with homologous DNA or protein sequences - analysis of gene expression by in situ hybridization or by northern blot analysis - analysis of protein expression by immunofluorescence staining

Genome analysis results

- Prokaryotic genomes ----- Eubacteria genomes - genome sizes vary - most circular, not all ex: E. coli. Not wasting space with genomes, uses everything. Outer circle genes are transcribed in clockwise direction, inner circle counter clockwise - importance of plasmids, some essential - gene density is high - not all operons contain genes from the same biochemical pathway which was unexpected - overlapping genes in eubacteria was shown but also not expected

Recombinant DNA method

- Purify target DNA - Digest target DNA with enzymes (restriction endonucleases) that cut DNA at specific sequences (known sequences) - Join these fragments with other carrier DNA molecules (vectors) - Results in RECOMBINANT DNA molecule - Recombinant DNA molecule is transferred into a host cell where recombinant DNA molecule replicates - Recombinant DNA passed on within population of host cells, making more copies - Cloned DNA recovered, analyzed etc. - Cloned DNA within host cell can be transcribed, mRNA translated, gene product isolated (eg. insulin) - hard to overestimate how this methodology has changed biological research

Forward genetics

- Random mutagenesis: generate one mutation per genome so that only one gene product is disrupted in each organism - Radiation tends to trigger null mutations due to chromosome breaks, deletions, translocations, and other major rearrangements - UV light, ethyl methanesulfonate (EMS), and nitrosoguanidine cause single base-pair changes or small deletions and insertions, usually resulting in conditional mutations -transposons are also used for mutagenesis

How did they actually do the influenza experiments?

- Random mutation of h5N1 virus until a key protein HA could bind to mammalian cell receptor (2 mutations) - Mutated HA gene was combined with seven genes from H1N1 pandemic virus - infecting ferrets with this hybrid virus caused additional mutations that made it highly transmissible -virus acquired third mutation one week after infection - next, during airborne passage between ferrets, virus had a fourth mutation which allowed for more effective spread of virus (it infected ferrets in neighboring cages but it was not highly pathogenic at this point and did not cause mortality)

Derivations of Basic PCR

- Reverse Transcriptase PCR (RT-PCR) -Amplify from RNA

Western blotting

- Screen proteins bound to a filter - Use antibody to the protein of interest as the probe

Gene knockout steps

- Start with cloned gene, altered to non-functionalize the gene, in this case by inserting the antibiotic neomycin resistance (neo') gene into the middle of it - add ES cells in culture (from agouti mice) - transform ES cells with targeting vector (random integration) - Grow transformed ES cells on medium containing neomycin and ganciclovir (if randomly integrated, the product of the thymidine kinase (tk) gene will preferentially phosphorylate ganciclovir (a nucleoside analog), instead of thymine, inhibiting replication --> dead cells) - There's homologous recombination too You find knockout gene after recombination with Gene X and the Neo disrupted gene - select surviving ES cell colonies -Test for presence of knockout gene - Grow up culture of knockout cells

Gene knockout goal (reverse genetics)

- Targeted gene knockouts involve deletion or disruption of a specific gene or protein to study the resultant phenotype - methods have been developed for targeted gene knockout in both yeast and mice - gene knockout goal is to replace homozygous wild type locus with homozygous mutant locus

Somatic Cell nuclear transfer method

- Transfer donor nuclei from Afghan hound somatic cells using SCNT - use mature oocytes removed from MIXED breed female dogs - remove nucleus and Barr bodies from oocytes - transfer donor somatic cell nucleus - fuse cells - implant fused embryos into surrogate mother - this method is REPRODUCTIVE CLONING - then use microsatellite analysis to prove genetic identity of any puppies produced

chromosome specific library

- Useful to study single chromosomes -Can separate single human chromosomes - Has been done for each human chromosome - Some chromosomes (eg. yeast) can be separated by pulsed field gel electrophoresis and isolated, then cloned

EACH PCR step, with temperatures

- You have dsDNA to be amplified (this is start of cycle 1) Step 1: Denature DNA (95C) 2: Anneal Primers (35-62C) 3: Extend Primers (68-72C) - product of first cycle is two new DNA molecules Cycle 2 Step 1 and 2 (Denature and anneal new primers) Step 3 (Extend Primers) Product of Cycle 2 is four new DNA molecules

Eukaryotic genome analysis results

- a mosaic of organization patterns - usually linear chromosomes - mitochondrial genomes present - chloroplast genomes in plants - genome size highly variable -low gene density - introns present - repetitive elements (up to 80% of genome in maize)

Increased gene diversity through gene recombination

- about less than 30000 genes in human genome

Steps of cDNA

- add dT primer - add reverse transcriptase - DNA synthesis continues - creates RNA-DNA helix - RNA digested by RNaseH -DNA pol I synthesizes complement DNA because of hairpin loop - add S1 nuclease to cleave hairpin, left with dsDNA - you can then clone ds cDNA -then have a library of EXPRESSED genes

Stem cell types

- adult stem cells: derived from regenerating tissue types - pluripotent: can be made to differentiate into more than one cell type -embryonic stem cells: derived from embryos - pluripotent: have much more ability to differentiate into many developmental pathways - totipotent = ability to differentiate into ANY cell type

human globin expression

- after birth, fetal hemoglobin gamma is shut down by BCL11A (a zinc finger transcription factor) - globin switching takes place after birth to beta globin, where it increases into adulthood - increase in BCL11A results in decrease in fetal hemoglobin gamma

cDNA library methods

- almost all mRNAs have polyA tail - method takes advantage of that - poly dT primer added to isolate mRNAs (this binds to poly A tail) - a complement synthesized by reverse transcriptase

Globin Duplication/Evolution

- ancestral duplication of an oxygen transport gene was about 800 mya --> 2 sister genes - 1 became modern day myoglobin (muscle oxygen carrier) - second became the ancestral globin gene - the second gene duplicated again about 500 mya

qPCR or real time PCR

- another derivation of PCR - quantitatively determine the amount of nucleic acid (to determine number of organisms) - qPCR is one of the main methods currently being used to screen for COVID-19 infections - if you can make specific primers for genome of interest you can quickly and specifically screen for sequence presence, and by extension organism's presence

antibodies

- antibodies (immunoglobulins) IgM, IgD, IgG, IgA, IgE found on plasma B cells - proteins produced by vertebrates act as a defense against infection - millions of different forms, each with a different binding site that specifically recognizes another molecule (antigen) - study antibody structure -specific antigen binding sites which are variable regions - under that is a hyper variable region and under that are constant regions - constant regions have an upper light chain and bottom heavy chain - 2 types of light chains (kappa or lambda) and 5 types of heavy chains - different combinations of chains create different Ig classes

Human ES cells

- are created and maintained in a similar fashion to mouse ES cells. They appear to be very similar in many respects to mouse ES stem cells.

transgenic mice available

- are created by microinjection of fertilized eggs with transgenic DNA - transgenic mice are used to determine the function of cloned human genes - gene knockout and targeted gene replacement are important genetic dissection techniques in mice

dog cloning challenges

- at ovulation oocyte is at first meiotic prophase - dog oocyte matures in oviduct over 2-3 days following ovulation - maturing the early ovulated oocytes in vitro was not successful

Reverse genetics

- begins with a cloned wild-type gene or protein - progresses to site-directed mutagenesis and phenotype analysis to understand gene function - a protein suspected of involvement in a particular phenotype can be purified and partially sequenced - an oligonucleotide probe designed from the amino acid sequence is used to probe a genomic or cDNA library to obtain the gene that encodes the protein

cosmos (another vector)

- can carry up to 50kb inserts (larger fragments) - cosmid: part phage, part plasmid - engineered vector - ligate fragments into cosmid, package into protein heads, infect hosts

Neuronal stem cells

- can form numerous tissues in vivo in mouse and chick embryos and are therefore pluripotent - in addition, blood isolated from umbilical chords is an excellent source of hematopoietic stem cells - it therefore appears that stem cells will offer a great deal of therapeutic value in medicine

Stem cells

- cell that stay in cell cycle, provide source of replenishing cells -e.g., epidermis, GI tract, mammary glands, lung, blood forming tissue, testis, ovary

lambda phage vector system

- central third of phage DNA expendable - cut out central third of lambda - leaves left arm, right arm, central region - isolate arms, recombine with DNA fragment of interest, & ligate together - foreign DNA inserted -after in vitro packaging, the recombinant viral particle is able to infect host cell carrying cloned foreign insert and replicate to form plaques

Ct

- cycle threshold values - defined as the number of cycle required for fluorescent signal to cross the threshold. -ct levels are inversely proportional to the amount of target nucleic acid in the same (the lower the Ct level the greater the amount of target nucleic acid in the sample) - strong vs weak positives

experimental design

- determine day of ovulation by following progesterone levels - harvest mature oocytes from female dogs Two portions of exp design ONE: Somatic Cell nuclear transfer: method used to produce the clone (somatic cell nucleus transferred into fertilized egg) TWO: micro satellite analysis to PROVE the dog is a clone

Development of modern techniques of Recombinant DNA Technology

- developed in 70s -revolutionary -first commercial approval was 1982 for bacterial produced human insulin -has led to explosion in this industry

Gene transfer to plant cell hosts

- done using bacterial plasmids as vectors

Epistasis analysis in forward genetics

- each mutant must have a slightly different phenotype - IF the phenotype of a double mutant organism homozygous for both mutations is the same as one of the single mutations, THEN that single mutation is likely to represent an earlier step in a pathway (shows which enzymes act earlier in the pathway)

good model organism traits

- easy to grow - short generation time - produce abundant progeny - readily mutagenized and crossed

Through gene knockout techniques, we can create mice that lack the leptin receptor (db-) (reverse genetics example)

- expression of the gene for the leptin receptor specifically in the neurons of the mice rescues the mutant phenotype in the db-/db- homozygous mutant mice

How do we use cloned DNA

- gene libraries - library screening, use labeled probe to screen library, chromosome walking - cloned fragment characterization: restriction mapping, nucleic acid blotting, DNA sequencing

success of CRISPR-cas

- has worked on all systems tested so far from drosophila to mice to humans - system has been used to simultaneously target multiple genes at the same time (5 have been targeted together) - two groups used these methods to disrupt most human protein-coding genes - reverses Huntington's disease in mice

Yeast artificial chromosomes (YACs)

- have telomeres, ori, and centromere - eukaryotic, acts like bacteria for growth and manipulation - extensively studied, entire genome seq. - to study function of eukaryotic proteins, need a eukaryotic system - yeast considered safe

Using YACs

- have telomeres, ori, and centromere - joined to selectable markers, cluster of RE sites - clone inserts 100 kb to 1 Mb - important vector for genome sequencing, eg. Human Genome Project

how do we obtain incredible diversity in immune system genes?

- immunoglobin gene subunit diversity - differential splicing to increase diversity - break and nibble mechanism to increase diversity result: incredible amount of diversity of immune system genes to produce antibodies for antigens

Uses of CRISPR-cas

- inactive genes of interest - off targeting issues can be minimized by variety of methods - transfect with minimal amount of plasmids - use mutant Cas9, which only cuts one strand, use a second guide DNA to the complementary strand and induce cut on other strand

continued lambda phage vector system

- introduce packaged ligated phage to host cell -phage replicate in host, make phage particles that contain inserts - form plaques on bacterial lawns

Targeted gene replacement (reverse genetics)

- involves in vivo substitution of a gene containing a specific site-directed mutation for a wild-type version of the gene

example of forward genetics methodology

- master plate with colonies growing at 23C -velvet pressed onto surface of master plate -velvet pressed onto two new plates (mutant grows at permissive temperature) - plates incubated at two different temperatures - conditional mutants: when incubated at restrictive temperature it does not grow - select the temperature-sensitive mutant colony from the master plate

Method of utilizing CRISPR-Cas

- method: only requires two items - ONE: Cas enzyme to cut DNA - TWO: RNA molecule (guide RNA = gRNA) - shorter version of CRISPR RNA, used to direct Cas to target and break DNA and inactivate gene

Mice as model systems

- mice are relevant and accessible model organisms for human disease studies - short generation times of 8 to 9 weeks - 8 or more offspring per mating - mice have similar body plans and stages of development as humans - similar genome size and number of chromosomes to humans - many human genes have homologs in mice - mice have drawbacks in comparison to yeast and drosophila ---- more difficult to grow, cross and mutagenize ---- large-scale genetic screens cannot be readily performed in mice

ALS in mice

- mice have been used as a model to study ALS - employs transgenic mice bearing a mutant human SOD1 gene - transgenic SOD mice develop symptoms of ALS similar to those seen in humans - the transgenic SOD mice also provide insights into potential therapies for ALS - in some cases, drug therapies have been tested in transgenic SOD mice first - drug therapies have moved to clinical trials for humans - gene therapy in transgenic SOD mice with Insulin Growth Factor (IGF-1) gene has shown promise

Genomics

- move to molecular methods (1980s), away from classical methods - genomic library clones pieced together - clones sequenced

mutant detection in forward genetics

- mutants detected using a genetic screen - visual examination of large numbers of mutagenized organisms looking for a mutant phenotype - dominant lethal mutations are usually not detectable in genetic screens -recessive mutations can be READILY DETECTED in yeast during haploid phase - observation of recessive mutations requires mating and crossing of the F1 progeny to be observed in obligately diploid organisms. - recessive lethal mutations isolated in haploid yeast as conditional mutations through replica plating

next vector system

- need to handle larger insert (10-15 kb) - bacterial virus (phage) vectors - eg., M13, lambda phage vectors

CRISPR Cas to the clinic

- novel technique to rescue mutant sickle cell phenotype - targets gene regulation pathway - results in adult expression of fetal hemoglobin - late 2019 results very promising - initial treatment was on a sickle cell patient and a Beta thalassemia patient

coronavirus genome structure

- one ssRNA - 30K bases

human genome results

- only less than 30000 coding genes (5% of genome) - 50% of genome repeat elements - gene clusters and gene deserts - wide range of intron numbers - bacterial derived genes in human genome - human genes tend to be large and contain introns - gene distribution is not even on chromosomes - duplicated regions found on some chromosomes - function of about 60% of genes are determined

Archaea genome analysis results

- organisms typically extremophiles - structurally similar to eubacteria, metabolically more similar to eukaryotes - have histone chromosomal proteins, chromosomes may be organized into chromatin - introns in tRNA genes

Methodology of crispr treatment

- patient undergoes chemotherapy (bisulfan) treatment to obliterate any remaining bone marrow hematopoietic stem cells - after chemotherapy, CRISPR treated hematopoietic stem cells are infused into patient blood stream and make their way to the bone marrow

DNA sequencing steps

- primer is bound to template strand - reaction ingredients are added (DNA pol, dATP, dCTP, dGTP, dTTP and a small amount of ddATP didieoxynucleotide which is the key to the procedure , it doesn't have a 3' OH so no extension of polymerization) - primers are extended and the chain terminates when a ddATP is inserted -newly synthesized strands recovered and get loaded on gel in "A" lane - you can do this with ddNTPs with fluorochromes - each fragment differs by one base in size, and a different color associated with each ddNTP - the next step is electrophoresis, imaging and data analysis

Genome analyses

- prokaryotic (eubacteria and archaea) and Eukaryotic

DNA microsatellites

- repeated DNA regions, can be di, tri, or tetra nucleotide repeats - single locus genes, an individual can have only 2 alleles at any one microsatellite locus - there are many different microsatellite loci in the genomes of many eukaryotes - flanked by unique DNA sequence region - are highly variable (key) in populations - very useful for ID purposes

Other uses of DNA sequencing

- screening for single nucleotide polymorphism (SNPs) - disease allele identification - phylogenetic and evolutionary studies ---- ribosomal RNAs have been extensively used for these sorts of studies

Selection in forward genetics

- selection to select for rather than screen for the desired mutant - goal of selection is to remove (by inhibition or killing) those organisms that do not display the relevant phenotype - leaves only the desired mutants in the population

Proteomics

- study of gene products - when, where produced - post translational modification - cellular localization - proteome: complete set of proteins expressed during a cell's lifetime

usages of CRISPR-cas

- system is now being modified, exploited and utilized in other systems, including eukaryotes - being used to specifically target and "knockout" a gene of interest -or being developed for gene correction and gene editing

results of globin duplications

- temporal expression of different genes - 3 alpha globin genes - zeta expressed only in embryo - alpha1 expressed in fetus - alpha2 expressed in adults - 5 beta globin genes - 3 genes expressed prior to birth - delta and beta globin expressed after birth

second duplication of globin

- the second duplication resulted in alpha and beta globin gene families - further duplication of alpha global results in 3 alpha genes on human chromosome 16 - further duplication of beta global results in 5 beta globin genes on chromosome 11

rest of CRISPR cas editing system for DNA

- the system does the rest -induces dsDNA break at target site - DNA repair mechanism cannot efficiently repair this damage and typically adds one or two nucleotides - frameshift mutation - if your target is early in the gene will typically make the product non-functional

cell-based cloning

- use a living organism to produce the replicates - usually using phage, bacterial, or yeasts hosts - plants are also now being used as a host

chromosome walking

- use series of probing experiments to move along region of chromosome - result of experiment 1 becomes the probe for experiment 2 - result of 2 becomes probe for 3, etc. - used after general position of the gene has been determined, e.g. from linkage analysis of disease and variable genetic markers in pedigree analysis - we examined this type of positional mapping in module 1 when we examined the physical location of the CMTX3 loci and narrowed it down to between 2 markers

Prove that Snuppy derived from the donor Afghan

- used microsatellite analysis using 8 dog microsatellites - proves beyond reasonable doubt that Snuppy's cells are derived from the donor dog

DNA microarrays

- used to examine the expression of thousands of genes simultaneously - microarrays can be used to compare the pattern of gene expression in two or more tissues - at different times during Development - in cells from normal vs diseased tissue

C. elegans genome

- very different from other eukaryotic genomes - 25% of genes in polycistronic operons - large number of introns - genes within introns ( a bunch of nested introns)

Cre-Lox knockouts

-Lox P is the target DNA sequence for Cre recombinase -Cre recombinase catalyzes recombination between two adjacent DNA sequences - a mouse carrying conditional (floxed) alleles for a gene of interest (GOI) is crossed with a Mouse carrying a Cre recombinase gene controlled by a tissue-specific promoter X -When crossed together, Cre "cuts out" gene of interest in target tissue - GOI "knocked out" in specific tissue based on promoter

Drosophila genetics methods

-Use balancer chromosomes with a dominant marker gene in Drosophila -Simplifies the genetics of this diploid organism -Also exploit P element transposons as a genetic tool to introduce cloned genes and to generate mutants

Nucleic acid blotting (southern blotting)

-Use the cloned fragment to further characterize gene of interest (narrow down location by screening RE digests with clone) -Screen related species -Also useful to identify number of copies of a particular sequence or gene

Two alternative libraries

-chromosome specific library -cDNA library

Gene libraries

-clone a large region or the whole genome -at least 1 copy of all sequences in host genome -performed using "host cloning" techniques -choose vector so that fewest # of clones required to get the whole genome

Genome evolution

-compare genomes to gain insight to genome evolution -bacteria: 3.5 billion years ago -eukaryotes: 1.4 billion years ago

Definition of clone

-identical organisms -identical cells -identical molecules -descended from a single ancestor

conservation of function

-many gene sequences are conserved from yeast to higher vertebrates -carry out similar functions in a wide range of organisms -allows fundamentals of metabolism, development, and disease to be studied in simple lab organisms -knowledge obtained applied to more complex eukaryotes

More forward genetics analysis

-observation of recessive lethal alleles in diploid organisms requires more intricate strategies - heterozygotes will not display recessive phenotype and homozygotes will die

First generation vectors

-pUC vectors (5-10kb) -derived from bacterial plasmids (extrachromosomal circular DNA naturally found in bacteria) - circular DNA molecule -replicates 500X in host -large # of RE sites (polylinker = multiple cloning site [MCS]) -selection system (ampicillin gene) -insert discrimination system (lacZ gene)

Cloning

-produces many identical copies -use for research -use commercially

A typical prokaryotic genome has

1 million base pairs of DNA, containing 1000 genes.

Basic PCR procedure

1) Denature target DNA 2) Anneal specific primers to flank DNA target of interest 3) Extend primers using Taq DNA polymerase Then repeat 25-40 times 25 cycles results in 10^6 increase in target

Predicted results

1. CRISPR CTX001 leads to decrease in BCL11A which leads to increase in fetal hemoglobin gamma 2. Fetal hemoglobin gamma rescues disease phenotype

Tissues examined

1. Donor dog, Afghan hound that was cloned 2. Snuppy, the cloned hound 3. The surrogate Lab mother

Gene therapy in transgenic SOD mice

1. Injection of muscles with Adeno-Associated Virus (AAV) containing IGF-1 gene 2. Virus taken up by neurons and transported to cell body 3. Neuron secretes IGF-1

first step of Crispr-Cas

1. Transfect cells with plasmids that express Cas enzyme and guide RNA (gRNA) -The Cas is Cas9 (from streptococcal bacteria), which can cut the DNA without help from other proteins. Makes a double stranded cut - gRNA, approx. 80 nucs. First 20 nucs complementary to target gene and the remaining 60 nucs form hairpin to help gRNA complex with Cas and facilitate binding of complex to target

Most of the bacterial genomes described in the text have fewer than

10,000 genes

Kary Mullis

1993 Nobel Prize Developed PCR method Parts known previously

human genome project

3.2 billion bases

Assume that a plasmid (circular) is 3200 base pairs in length and has restriction sites at the following locations: 400, 700, 1400, 2600. Give the expected sizes of the restriction fragments following complete digestion.

300, 700, 1000, 1200

The haploid human genome contains about 3 × 109 nucleotides. On average, how many DNA fragments would be produced if this DNA was digested with restriction enzyme PstI (a 6-base cutter)? RsaI (a 4-base cutter)? How often would an 8-base cutter cleave?

4 base cutter = (1/4)^4 = 1/256bp gets cut (3x10^9)/256 = 11,718,750 cuts6 base cutter = (1/4)^6 = 2.44e-4 ~ 1/5000bp gets cut (3e9/5000) = 600,000 cuts8 base cutter = (1/4)^8 = 1e-5 ~1/100,000 gets cut (3e9/100000) = 30,000 cuts

The full-length (i.e., containing the entire protein-coding region) cDNA for a specific eukaryotic gene in humans is 1500 nucleotides long. You screen a pig genomic library with this cDNA and isolate two genomic clones of different lengths. Both clones are sequenced and found to be 1900 and 2100 nucleotides long from start codon to stop codon. Screening of genomic libraries of several other organisms reveals that all of them contain only one genomic clone -- pigs seem to be the exception to the rule here. What evolutionary events might have led to the presence of two genomic clones in pigs, and the discrepancies in their length compared to the cDNA probe? How is this representative of a general type of occurrence in molecular genetic evolution?

50% credit: There was likely a duplication of this gene in pigs.25% credit: After duplication, the gene has diverged.25% credit: Evolutionary divergence tends to follow gene duplications, as mutations in one gene are no longer selected against as strongly (the presence of a "back up" copy of the gene means the individual will generally have at least one functional copy of the gene product).

What is a cDNA molecule?

A cDNA molecule is a DNA copy of an RNA molecule. Or complementary DNA

In the genetic map of the human genome, one map unit is approximately 850,000 bp. For the genome of the eukaryotic yeast Saccharomyces cerevisiae, one map unit is approximately 3000 bp. What is a map unit, and why is it so different in these two different types of organisms?

A map unit is the amount of measured recombination between two linked points in a genome.Because one map unit in humans is many more base pairs than in yeast, the amount of homologous recombination per DNA length must be lower in humans than in Saccharomyces cerevisiae.

Describe the three basic components of a typical plasmid cloning vector and the reason/use for those plasmid vector components.

A typical plasmid vector will have the following components:-Polylinker: First, there will be a multiple cloning site OR polylinker that is "cut" with specific restriction enzymes. The same restriction enzyme can then be used to "cut" templated DNA. When the cut vector and template are mixed together the complementary single stranded regions will anneal to one another and this complementarity is covalently linked using DNA ligase. Once in the vector, the vector is transferred into a bacterial host, where is multiplied.-Antibiotic resistance marker: The vector also contains antibiotic resistance genes so that when growing in the presence of antibiotic, only those bacteria with the vector will be able to grow.-Selectable marker: Lastly, the vector will contain a selectable marker, eg. the lacZ gene. The polylinker is located within this gene, when a template is ligated within the lacZ gene it is made nonfunctional. In the presence of a colorimetric indicator, such as X-gal, which is broken down into a blue byproduct by lacZ, the presence of a template fragment in the plasmid vector is visualized by white bacterial colonies. These colonies can then be chosen for further analysis.

In the context of recombinant DNA technology, what is meant by the term vector?

A vector is a vehicle to carry recombinant DNA molecules into the host cells where independent replication can occur. Most common vectors are plasmids, bacteriophages, and cosmids.

Stem cells in medicine

ALS - fetal stem cell treatment of ALS

You determine that you have only three copies left of an important DNA fragment, so you decide to amplify it. Using flanking primers, how many PCR cycles would you have to run to generate over one billion (10^9) copies of the fragment?

Accept anywhere from 28 to 30 cycles as a correct answer or the following equation 3 x (2^n) => than 1 billion)

Clones can be of a cell, an organism, or a molecule. What characteristic do they all have in common?

All are derived from a single ancestor

What do PCR, reverse transcription, and dideoxy DNA sequencing all have in common?

All produce DNA chains as a product.

SOD1 gene

Amyotrophic lateral sclerosis (ALS) genetic associations - superoxide dismutatse 1 Dominant mutant found in 20% if familial ALS disease cases - SOD1 is an antioxidant enzyme that converts free radicals, which protects sells from these damaging byproducts of respiration - single amino acid sub from glycogen to alanine at aa position 93 (G93A) - cloned human G93A used for transgenic mice studies

A section of a genome is cut with three enzymes: A, B, and C. Cutting with A and B yields a 10-kb fragment. Cutting with B and C yields a 2-kb fragment. What is the expected result from a digest with A and C, if the C site lies in between the A and B sites?

An 8-kb fragment

What is a transgenic organism?

An organism that contains genes from other organisms

Bioinformatics

Analyze and store vast amounts of data - visualize data -access data - data mining - many companies have developed bioinformatic software

Other studied model organisms

Asoergillus nidulans, C. elegans, Danio rerio (zebrafish), arabidopsis thaliana (plant model organism)

Under ideal conditions, how many copies of all the sequences of the host genome should be represented in a genomic library? Why?

At least one should be represented. Typically, library construction includes a several-fold greater number of clones than necessary for one representative of each fragment in order to increase the likelihood of cloning difficult fragments and stochastic loss.

Molecular biologists rely on many, often sophisticated, techniques to pursue their discipline. One may list ultracentrifugation, electron microscopy, X-ray diffraction, electrophoresis, and computer interfacing as fundamental. Model organisms provide the raw materials for study. List four "organisms" (or organismic groups) often used by molecular biologists and describe a major advantage of each group to the molecular biologist. We might consider these as "model organisms" of the molecular biologist.

Bacteriophage: relatively simple, short generation time. Bacteria: relatively simple, short generation time, simple growth requirements. Yeast: relatively simple for a eukaryote, short generation time, simple growth requirements. Drosophila: relatively simple to culture, extensive genetic and developmental information available, "giant" polytene (salivary gland) chromosomes.

ChIP-chip

Chromatin-ImmunoPrecipitation-microaray chip -genome-wide screen for transcription factor binding sites using microarrays -POI = protein of interest - stick it to antibody (cross link and shear) - IP - immunoprecipation. Isolated POIs to antibodies on matrix -purify, amplify, and label - now have isolated, and fluorescently tagged, DNA that bound POI - genomic DNA microarray representing whole genome in small fragments bound to substrate

Genome sequencing methods

Clone by clone and shotgun method

CRISPR-Cas DNA editing system

Clustered Regularly Interspaced Short Palindromic Repeats -found in 40% of bacteria and 90% of archaebacteria examined -these are loci that express small RNAs that match sequences found in the genomes of invading bacterial viruses (bacteriophage) -when infected these CRISPR RNAs binds the viral genome through complementarity and brings associated protein Cas - Cas enzymes are nucleases that cut the viral DNA which ENDS the virus

Recombinant DNA

Combination of DNA molecules not naturally found together - methods derived from nucleic acid biochemistry

What is comparative genomics? How does its study contribute to our understanding of genetics?

Comparative genomics is a relatively new field involved in identifying similarities and differences in organization and gene content among the genomes of different organisms. Such studies are important for studying the genetic relatedness of species and for identifying gene families.

Emerging DNA-Editing technology

Crispr/cas

A human gene with a disease phenotype is going to be mapped by positional cloning. Which would be the most useful for this task?

Data about the inheritance of SNP markers in families with the disease

Fluorescent Sanger dideoxy sequencing methods uses what method to discriminate between the 4 different nucleotides?

Different fluorochromes attached to the four different ddNTPs.

Explain why the genetic map distance between two genes on the same chromosome may be inconsistent with the physical map distance. E.g., for three loci A, B, and C, on the same chromosome, explain why the genetic distance might be A-[20 centimorgans]-B-[20 cM]-C, while the physical distance might be A-[200 kilobases]-B-[100 kb]-C.

Different regions of the chromosome will be more prone to recombination than others.

restriction endonucleases (RE)

Each RE recognizes a specific sequence of DNA (recognition site), which is a palindrome, same sequence 5' -> 3' on opposite strands - makes complimentary tails - specificity

Restriction endonucleases

Each RE recognizes a specific sequence of DNA (recognition site), which is a palindrome, same sequence 5' -> 3' on opposite strands.

The PCR (polymerase chain reaction) protocol that is currently used in laboratories was facilitated by the discovery of a bacterium called Thermus aquaticus in a hot spring inside Yellowstone National Park, Wyoming. This organism contains a heat-stable form of DNA polymerase known as Taq polymerase, which continues to function even after it has been heated to 95 degrees C. Why would such a heat-stable polymerase be beneficial in PCR?

Each cycle includes a "hot" denaturation phase (95oC), which separates the hydrogen bonds that hold the strands of the template DNA together.

As a model system, what are three of the advantages of the mouse as a model system?

Easy to grow • Short generation time • Produce abundant progeny • Readily mutagenized and crossed. Mice have similar body plans and stages of development as humans • Similar genome size and number of chromosomes to humans

Mario Capecchi, Sir Martin Evans, and Oliver Smithies recently won a Nobel Prize for gene targeting (gene knockouts) in mice. Describe the steps involved in creating a knockout mouse.

Embryonic stem (ES) cells heterozygous for a knockout mutation in a gene of interest (X) and homozygous for a marker gene (here, black coat color) are transplanted into the blastocoel cavity of 4.5-day embryos that are homozygous for an alternate marker (here, white coat color). The early embryos then are implanted into a pseudopregnant female. Some of the resulting progeny are chimeras, indicated by their black and white coats. Chimeric mice then are backcrossed to white mice; black progeny from this mating have ES-derived cells in their germ line. By isolating DNA from a small amount of tail tissue, it is possible to identify black mice heterozygous for the knockout allele. Intercrossing of these black mice produces individuals homozygous for the disrupted allele, that is, knockout mice.

Double digest mapping problem

Enzyme 1: 350, 950 (sizes of fragments, not location of a 1300 size piece of DNA) Enzyme 2: 200, 1100 Enzyme 1 and 2: 150, 200, 950

Present an overview of the gene organization in large-genome plants. What general level of gene density do plant genomes normally exhibit? What is the composition of the non-gene portions of the DNA?

Generally genome divided into 1. highly repetetive sequence (10%): It is non gene portion, contains 1. Tandem repeats:. 2.Dispersed repeats: 2. moderatly repetative sequence (20%) : Gene families 3. non repetative sequence(70%): Almost all structural genes.

Functional complementation

Genes identified by mutational analysis in yeast cloned through functional complementation - method that allows you to test all cloned yeast genes to determine which one will rescue the mutant phenotype, this determining which gene has acquired the mutation. - this approach cannot be used in drosophila, mice, or humans for example ---- map based or positional cloning used to narrow down location of mutant gene ---- chromosome walking then used to clone a gene identified by a mutant phenotype

Crossing over is often reduced around centromeric regions of chromosomes. If you were trying to construct a genetic map of two linked marker loci in this region, what result might you obtain and why? How would the genetic map correspond to the physical map?

Genes mapped based on recombination will appear to be very close together in centromeric regions due to low rates of recombination. Distances between the same genes on the physical map may be much greater when compared to other regions of the chromosome.

Clone-by-clone method

Genetic map of markers, such as RFLPs, STSs spaced about 1 Mb apart. This map is derived from recombination studies. RFLP: Restriction Fragment Length Polymorphism: occurrences and location of RE recognition known STS: Sequence Tagged Site, 200-500bp site that has a single occurrence in the genome and location is known Physical map with RFLPs, STSs showing order, physical distance of markers. Markers spaced about 100,000 bp apart. Set of overlapping ordered clones each covering 0.5-1.0 Mb. Each overlapping clone will be sequenced, sequences assembled into genomic sequence of 3.2 x 10^9 nucleotides

Model organisms

Geneticists use model organisms that are genetically tractable

steps of chromosome walking

Get initial adjacent sequence (B) - Subclone fragment, use to probe for overlapping clone - subclone, re- probe (C) - goes on and on until the target gene is cloned (F)

LacZ insert identification method

Grow bacteria with X-gal • When intact, LacZ gene product results in blue colonies by metabolizing X-gal • However, if a DNA fragment is inserted in polylinker region, LacZ gene is inactivated • Result, no X-gal metabolism==>WHITE COLONIES

Human genome example

Human genome: 3bil bases of DNA Assume P = .pp (probability of getting a particular sequence = 99%) - use lambda vector (17 kb per vector), f = 17000/ (3 x 10^9) = 5.7 x 10^-6 N = 807894 That is number of lambda clones to cover the human genome at 1 fold (1 X) coverage

Explain why the greatest diversity of human SNPs is found among African people.

Humans are thought to have first evolved in Africa. This means that any distinct population of humans (however defined) arose from a subset of the African population that became geographically isolated. Thus, any SNPs in this population arose from precursors that were already present in the African population, and another branch of those ancestral SNPs' descendents is likely still extant in the African population. Basically, for any SNP "family" in a distinct human Population X, the African population probably has a SNP family very similar to that one, plus several others with no clear analogue in Population X.

In the polymerase chain reaction, what is the purpose of the initial high temperature? What is the purpose of cooling in the second step?

In PCR, the initial high temperature serves to denature the double stranded DNA into single stranded DNA. Cooling in the second step allows for the annealing or hybridization of the primer to the DNA strand, flanking either side of the target DNA.

Nucleic acid blotting is commonly used in molecular biology. Two types, Southern blots and northern blots, involve gel electrophoresis and a filter, which holds the nucleic acid. Briefly describe the procedure of "blotting" in this context and differentiate between Southern and northern blots.

In a Southern blot the DNA to be "probed" is cut with a restriction enzyme(s); then the fragments are separated by gel electrophoresis. Alkali treatment of the gel denatures the DNA, which is then "blotted" onto the filter (nylon or nitrocellulose). A labeled probe (RNA or DNA) is then hybridized to complementary fragments on the filter. In a northern blot, RNA is separated in the gel and "probed" with the labeled DNA.

Gene knockout in mice

Inject ES cells into blastocyst - black hair is recessive to agouti - black hair color allele = b - wild type agouti allele = B - generation of mice has black and agouti hair - chimera may have agouti germ cells - mate chimera to black mouse (bb) - some offspring will be black hair colored mice, not of interest because black hair is recessive. These mice did not get a germ cell derived from the agouti ES or they would not be black haired, they are b/b with respect to hair color and +/+ with respect to gene of interest - if mice are agouti colored, they are B/b for hair color, therefore the gamete from the chimeric mouse derived from ES, so it also has the ko in the ES, and is presumably heterozygous +/ ko for gene X. - The gene locus is +/ko - use PCR to test for presence of the ko allele

The first commercial production of what human enzyme led to the explosion of the biotechnology industry?

Insulin

Which of the below are not steps in the production of genome sequence maps:

Isolate whole chromosomes

What might be a reasonable function of restriction endonucleases in a bacterium, distinct from their use by molecular biologists?

Isolated from bacteria, restriction endonucleases restrict or prevent viral infection by degrading the invading nucleic acid of the virus.

The human genome contains approximately 20,000 protein-coding genes, yet has the capacity to produce several hundred thousand gene products. What can account for the vast difference in gene number and product number?

It is estimated that 40 to 60 percent of human genes produce more than one protein by alternative splicing.

Shotgun method

Method: Sequence all of clones and let computers figure out where they overlap and then assemble sequence Steps - have bacterial chromosome and fragment it by sonication - with the sonicated fragments, prepare a clone library - the clones from the clone library are sequenced - the sequence is assembled on a computer with compiler software

Number of clones can be calculated

N = ln(1-P)/ln(1-f) N = number of clones required P = probability of recovering a particular sequence f = fraction of the genome in each clone

NCBI website

National center for biotechnology information -repository of sequence/annotation data - genome sequence databases - protein databases - bioinformatic tools, eg BLAST for sequence similarity searches

What are Northern analyses used for? Describe the steps involved in performing a Northern analysis, and describe how levels of gene expression are determined.

Northern blots are used to detect/compare/study RNA in a sample. Steps: (1) RNA is extracted from tissue, cells etc. (2) Gel electrophoresis separates RNA molecules based on size (3) RNA is "blotted" (transferred) from the gel onto a membrane. (4) RNA is fixed to the membrane and hybridized with a probe (probe is labeled radioactively or with enzymes for chemiluminesence) (5) Visualization of RNA using X-ray film or phosphoimager screen. RNA levels can be determined and quantified by the darkness of the bands in the image.

In what way are specific DNA sequences of the template amplified in the polymerase chain reaction? In other words, how does one target the target?

Oligonucleotide primers associate by hydrogen bonding to specific sections; primers are then extended

Designed region of Sligo complementary to gene target on the gRNA

Only requirement of oligo design is that it must bind to DNA region that has sequence -NCC at the 5' end (this is sequence found in bacteria, its called the Protospacer Adjacent Motif [PAM]) - gRNAs bind best to DNA where opposite strand contains a sequence -NGG -computer program can help design oligo for particular targets and minimize off-targeting

How do we monitor/test for H5N1?

PCR

Taq polymerase

PCR

type of cell free cloning

PCR

How are pseudogenes formed?

Point mutations, deletions, and duplications.

What is meant by the term pseudogene?

Pseudogenes are nonfunctional versions of genes that resemble other gene sequences but that contain significant nucleotide substitutions, deletions, and duplications that prevent their expression. Pseudogenes are designated by the prefix (psi).

What is recombinant DNA technology? What are the safety issues related to recombinant DNA technology?

Recombinant DNA technology refers to the creation of new combinations of DNA molecules that are not normally found in nature. Safety issues generally center around the creation and release (accidental or intentional) of genetically engineered organisms that are a threat to human health or animals and plants in the environment. Many organisms that are "genetically engineered" carry genes for antibiotic resistance.

Methodology

Remove hematopoietic (blood) stem cells from patient - electroporate CRISPR CTX001 targeting BCL11A into hematopoietic stem cells in vitro

Electrophoresis separates DNA fragments of different sizes, but this technique does not indicate which of the fragments contains the DNA piece of interest. This problem is solved by

Removing the bands from the gel and hybridizing them with a known strand of DNA complementary to the gene of interest

nested or semi-nested PCR

Second PCR amplification using one internal primer (semi-nested) or two internal primers (nested) another derivation of basic PCR

Assume that you have cut λ DNA with the restriction enzyme HindIII. You separate the fragments on an agarose gel and stain the DNA with ethidium bromide. You notice that the intensity of the stain is less in the bands that have migrated closer to the "+" pole. Give an explanation for this finding.

Since the smaller fragments migrate toward the "+" pole, away from the origin, they bind less stain than the larger fragments near the origin.

Immune response

Somatic recombination occurs in maturing B cells - recombination activating gene produces enzyme (RAG) that cuts dsDNA - RAG evolved from a transposes enzyme derived from a transposable element - each mature B cell makes ONE type of light chain (kappa or lambda) and ONE type of heavy chain - an antigen stimulates a particular B cell with an antibody for that antigen - produces population of plasma cells with antibody for that particular antigen

Suppressor and enhancer mutations in forward genetics

Suppressor mutations rescue the original mutant phenotype Enhancer mutations increase the intensity of the mutant phenotype - can pinpoint additional genes in a pathway that may not have been identified in original genetic screens

Cloning of PCR products

T/A cloning, derived from plasmid vector

Some restriction enzymes cleave DNA in such a manner as to produce blunt ends. Most often ligation of blunt end fragments is enhanced by the use of the enzyme terminal deoxynucleotidyl transferase. Why?

Terminal deoxynucleotidyl transferase extends single-stranded ends by the addition of nucleotide tails.

What is meant by the designation EcoRI? Do not simply state the type of macromolecule designated by EcoRI, but explain why it is so named.

The Eco part of the enzyme's name originates from the species from which it was isolated, while the R represents the particular strain, in this case RY13.

What is the purpose of an antibiotic resistance gene in a plasmid cloning vector?

The antibiotic resistant gene provides a selectable marker for cells.

What two factors contribute significantly to the wide ranges of genome size among eukaryotes?

The presence of introns and repetitive sequences

You have cut DNA from source A with restriction enzyme #1 and you have cut DNA from source B with restriction enzyme #2. Both of these restriction enzymes leave a 4 base single stranded overhang. You want to ligate these restricted fragments together. What must be true for this to be successful?

The single stranded overhangs must be complementary to one another and DNA ligase must be present to seal the fragments together.

Describe the organization of the α-globin gene cluster in humans. Roughly how large is this cluster? How many genes are present? Briefly describe these genes.

The α-group spans more than 30 kb and contains three genes: zeta and two copies of the alpha gene. In addition, two nonfunctional pseudogenes are in the group. Most of the DNA in this region consists of intergenic spacer DNA.

Take home message

There is much to learn as we sequence multiple genomes and move away from heavily studied "MODEL" organisms. Results may challenge our models of generalization of gene organization, mechanisms, etc.

Why are telomeres and centromeres particularly difficult to sequence?

They consist of highly repetitive DNA, and so strand slippage issues can confuse the determination of a consensus sequence.

What is the function of dideoxynucleotides in Sanger DNA sequencing?

They stop synthesis at a specific site, so the base at that site can be determined.

This term refers to the work undertaken by large teams of researchers who, through a concerted effort, clone and sequence the DNA of a particular organism.

This term refers to the work undertaken by large teams of researchers who, through a concerted effort, clone and sequence the DNA of a particular organism.

What is the purpose of a cDNA library?

To produce a library of expressed genes.

What term is used to refer to the process in which DNA can be introduced into host bacterial cells?

Transformation

Name 2 methods that are used to produce mutations in a forward genetics approach.

UV light EMS Nitrosoguanidine

centromere

YAC

What is bioinformatics?

a method that uses very large national and international databases to access and work with sequence information

The lungfish Protopterus aethiopicus has a genome 38 times larger than that of humans. Most of the DNA in this species is noncoding repetitive DNA. How could you create a library of clones that would let you compare just the genes in the lungfish to the genes in humans?

You could generate cDNA libraries and compare the transcribed regions of the genome.

PCR is

a technique for amplifying DNA sequences in vitro

Plasmids are important in biotechnology because they are

a vehicle for the insertion of foreign genes into bacteria

vector requirements

ability to replicate itself and the DNA fragment it carries - RE sites in vector - selectable marker (eg. antibiotic resistance) to distinguish hosts with and without vector -easy to recover vector from host cells

sickle cell disease

autosomal recessive disorder that can result in a debilitating phenotype in individuals homozygous for this allele - vaso-occlusive crisis: blockage of blood vessels by sickled cells leading to extreme pain and potential irreversible organ damage - migration change due to a single amino acid change (neg charged glu to uncharged valine)

Write the letter all of the following statements that are NOT true. a. Coding sequences for gene products can be isolated from cDNA libraries. b. Antibodies are used for Northern blot analysis. c. VNTRs are highly conserved in human populations. d. PCR amplification generates large numbers of linear DNA fragments. e. RNA molecules can be used as hybridization probes in Southern blot analysis.

b,c

A principal problem with inserting an unmodified mammalian gene into a bacterial plasmid, and then getting that gene expressed in bacteria, is that

bacteria cannot remove eukaryotic introns.

For a physical map of a chromosome, distances are measured in units of

base pairs

What are three key differences between a genomic and a cDNA library?

cDNA lib. represents only transcribed regions of the genome; all genes equally represented in genomic library while cDNA library reflects the level of expression of a gene in a particular cell type or tissue ; cDNA library contained only sequences found in the mature mRNA - introns are removed

Name the two strategic methods that scientists are using to sequence genomes.

clone by clone and shotgun sequencing

plasmid

cloning vector

The Human Genome Project, which got under way in 1990, is an international effort to

construct a physical map of the 3.3 billion base pairs in the human genome.

A set of overlapping DNA fragments that form a contiguous stretch of DNA is called a _________.

contain

A bacterial polycistronic transcription unit is one that

contains information for more than one protein product.

Lymphomas

different types depending on stage of B cell that cancer develops

One of the primary reasons for generating a large number of clones in a eukaryotic genomic library is that

each vector can take up only a relatively small fraction of the eukaryotic DNA.

gold standard of stem cells

embryonic stem cells

Gene transfer to mammalian cell hosts

encapsulation or endocytosis to transfer DNA to mammalian cell hosts micromanipulation

nuclear pore complex

example of genomics combined with proteomics to determine structure and function -genomic information determined genes involved in pore complex - proteomics to determine molecular architecture of pore complex - next: study interaction to determine what interacts with nuclear pore complex

A BLAST search is done to

find similar gene or protein sequences.

Gene duplication

important in origin and evolution of eukaryotic genomes - increases genetic diversity - results in multigenerational families - arise in unequal crossover - arise in replication errors - regions are duplicated in bulk

future work

improve methodology to increase efficiency - apply methods developed to endangered or threatened or rare species

Of what advantage is it to have a polylinker region (multiple unique restriction sites) embedded in the lacZ component in the pUC series of plasmids?

insert of DNA in polylinker inactivates lacZ component and allows identification of recomb plasmids under proper genetic and environmental conditions

Kinome

interaction of kinase proteins, important in cancer research (kinases phosphorylate and are involved in signal transduction + cell cycle pathways)

One major difference between prokaryotic and eukaryotic genes is that eukaryotic genes can contain internal sequences, called ________, that get removed in the mature message.

introns

Targeted gene knockouts (reverse genetics)

involve deletion or disruption of a specific gene or protein to study the resultant phenotype

Protein-DNA interactions

mapped and used to identify genes that are regulated by DNA-binding proteins

Another word for a "DNA chip" (microscopic spots of oligonucleotides bound to glass that can be fluorescently labelled to identify levels of expression).

microarray

Some vectors such as pUC18 and others of the pUC series contain a large number of restriction enzyme sites clustered in one region. What term is given to this advantageous arrangement of restriction sites?

multiple cloning site

Ti

naturally occurring plasmid in bacteria (R r) When the Ti plasmid bacteria infects plant cells, a segment of the plasmid (T-DNA) is transformed into the host genome It is used to experimentally infect plant cells and transform a Gene of Interest (GOI) INTO the host plant genome if it is in the T-DNA region - ex: making plants herbicide resistant

stem cells

normal cells don't divide and are differentiated - stem cells are not fully differentiated and give rise to cells that are unlike themselves - they are also capable if self-renewal

In general, the organization of genes in bacteria is different from that in eukaryotes. In E. coli, approximately 27 percent of all genes are organized into contiguous, functionally related units containing multiple genes under coordinate control that are transcribed as a single unit. Such contiguous gene families are called

operons

Maturation of B cells

order from first to last pluripotent stem cell lymphoid stem cell progenitor B cell Precursor B cell Immature B cell Mature B cell

List the three basic components required for a bacterial cloning vector and briefly describe the purpose of each.

origin of replication for propagation in the host; selectable marker like Amp resistance; polylinker or unique restriction enzyme sites to facilitate cloning

A map of the order, overlap, and orientation of physically isolated pieces of the genome.

physical map

Transgenic

plants or animals carrying a foreign gene are transgenic

interactome

protein interactions with each other - description of protein-protein interactions within an organism - aids in understanding pathways and interactions - may provide insight for therapeutic interruption of pathway in disease treatment

SELF EVALUATIONS

question bank

What is the name of the process by which bacterial colonies (cells) are transferred from one agar plate to another, maintaining the same spatial pattern?

replica plating

Which of the following are the important proteins needed for cloning a eukaryotic gene into a bacterial plasmid?

restriction enzymes specific for the target genes DNA ligase

Restriction endonucleases are especially useful if they generate "sticky" ends. What makes an end sticky?

single-stranded complementary tails

During gel electrophoresis, __ will migrate more rapidly than __.

small DNA fragments; large DNA fragments

Over the years, sophisticated plasmid vectors have been developed for use in recombinant DNA technology. Describe the useful features that have been introduced in these vectors.

small size to allow large inserts high copy number large numbers of unique restriction sites (polylinkers) variety of selection schemes (pigmented colonies, antibiotic resistance)

Compared with eukaryotic chromosomes, bacterial chromosomes are

small, with high gene density.

Conclusion

snuppy is a clone - method worked, still very inefficient - different animals (mammals) present different challenges due to their particular biology - eventually, snuppy was able to be a father via in vitro fertilization

What is a concise definition of proteomics?

the process of defining the complete set of proteins encoded by a genome

When two proteins show a 50 to 70 percent match in amino acid sequence, it is likely that

the two proteins share a common ancestry.

Assume that one conducted a typical cloning experiment using pUC18, transformed an appropriate host bacterial strain (one carrying the lacZ complementing region), and plated the bacteria on an appropriate X-gal medium. Blue and white colonies appeared. Which of the two types of colonies, blue or white, would most likely contain the recombinant pUC18? Explain your answer.

the white colonies because of insertional inactivation of the lacZ component

If a restriction enzyme cuts a circular DNA into three fragments, how many restriction sites are there in the DNA?

three

Embryonic stem cells

totipotent - can make all tissues of a mouse, including germ cells - we can create mice that are made up completely from ES cells - can be differentiated to different cell types in vitro

What term is used to refer to the process in which DNA can be introduced into host bacterial cells?

transformation

vectors

used to carry DNA fragments - used as a way in (a vector) to a host cell where the vector and fragment can be replicated

complementation in forward genetics

used to determine whether two mutations are the same gene or represent mutations in separate genes - complementation analysis is performed by mating two homozygous mutant strains - examine the F1 progeny for the wild type and mutant phenotypes -technique only works for recessive mutations

The difference between a genetic screening experiment and a selection experiment is that a screening experiment involves ________, whereas a selection experiment creates conditions that ________ irrelevant organisms.

visual examination, eliminate

PCR advantages

• FAST, no cell based cloning required • done by programmable machine, essentially a glorified heating/cooling block • PCR is VERY sensitive, can work with only a few targets • great for forensics, degraded DNA, etc

Yeast Chromosome III results

• Found half the genes on yeast chromosome III were unique • Big surprise given how genetically well characterized yeast is • Also shows that sequencing of entire chromosomes or genomes can reveal more info than observed by classical mutational analysis

PCR limitations

• PCR is VERY sensitive, can lead to false positives due to contaminating DNA • standard PCR requires knowledge of DNA sequences surrounding region of interest

Yeast as a model organism

• Yeast (Saccharomyces cerevisiae) is a favored model organism • Alternating haploid and diploid phases in the yeast life cycle are particularly useful for genetic analysis • Yeast genome has been completely sequenced • Wide variety of mutants and deletion strains are available.