BIMS 320 Final Overview
What are phage vectors?
- Amongst earliest vectors used in addition to plasmids - include genetically modified strands of bacteriophage - have multiple cloning site - Carry up to 45 kb of cloned DNA
Blue v white colonies (recombinant??)
- Blue = cells with functional lacS gene carrying a nonrecombinant plasmid - White: bacterial cells with recombinant plasmid - Nontransformed cells did not grow due to presence of ampicillin in plating medium
What are the applicatins of bioinformatics?
- Comparing DNA sequence - Identify genes in genomic DNA sequence - Finding gene-regulatory regions(promoters and enhancers) - Identify telomeric sequenes - Deducing evolutionary relationships between genes
What is a homologous gene?
- Genes that are evolutionary related - similarity searches are able to identify homologous genes
What is the main difference between genomic and cDNA libraries?
- In genomic libraries all sequences-genic and intergenic- are represented approximately equally - In Embryonic cDNA clomes represent only genes 2 and 3 - In Eye cDNA clones represent only genes 1 and 3 - Both cDNAs have frequencies that reflect levels of gene expression and found in mature mRNA(no intron or intergenic sequence)
What is the result of the blue-white screening mechanism and the components it uses? (Slides 20-21)
- It was used to identify cell containing recombinant and nonrecombinant DNA 1: Agar plates contain X-gal(Analog of lactose - substrate for B-galactosidase) 2: When X-gal is cleaved by enzyme, it turns blue
What is gene knockout?
- Loss of function mutation - DIsrupt or eliminate specific gene/genes and see "what happens" - Knockout (KO) mie have revolutionized research
How are SNPS, INDELs, and CNVs a part of WGS?
- Single nucleotide polymorphism - Insertions and deletions - Copy-number variant is a repeat section of genome where each repea is greater than 1 kb long
What is a restriction map and how can they be used?
- Use data from them to generate maps of DNA sequences - provide information for further experiments
What is a probe?
- Used to screen library and recover clones of specific gene, muct be labeled or tagged - any DNA or RNA sequence complementary to target gene of sequence being identified
What is whole-genome sequencing?
- a process that determines an entire genome's sequence - most widely used strategy for sequencing and assembling an entire genome
What is the process of a de novo assembly? (ie., making libraries, sequencing, contigs, alignment, etc.)
- assembling a genome from scratch by overlapping DNA sequences into entire chromosomes 1: Reads 2: assemble 3: Contig 4: map reads to contigs 5: Contig1 and Contig2 6: assemble contigs to scaffolds 7: Scaffold 8: Gap filling
what are the areas of inquiry of genomics?
- structural genomics - functional genomics - comparative genomics
What is gene targeting?
Target specific allele, locus, or base sequence and learn its function on gene of interest
what is the insert size capacity of a BAC?
100-200 kb
The amino acid sequence of insulin was determined in the ________**
1950s
What was the main problem of repetitive DNA and how does paired-end sequencing remove this problem? (see insert of image slide 17-18)
1: >50% of mammalian genomes are comprised of repetitive DNA elements; LINES(long interspersed elements: 6-7 kb) have 500000 in human genomes(17%); SINES(short interspersed elements: 300-bp) have 1.6M in human genome (13%) 2: Endogenous retroviruses: XXXX 8in human genome(8%) 3: Satellite repeats 4: Telomeres - If the paired-end sequences flank a repetitive element, they can be used in assembling scaffold, a set of contigs physically linked by paired-end sequences that contain the repetitive element
Steps in PCR with temperatures (also review from older material; slide 36)
1: Denaturation: dsDNA is denatured into single strands (92-95 C) 2: Hybirdization/Annealing primers: Primers bind to ssDNA; starting point for DNA polymerase to ythesize new DNA trands(45-65 C) 3: Extension primers: DNA polymerase synthesizes DNA strands(65-75 C)
What are the 4 consequences of mitochondrial inheritance in mammals?
1: Predication of mitochondrial mutation inheritance can be based on mother's genotype 2: Maternal lineage of organisms can be examined 3: Maternal history of species can be interpreted 4: Human genetic diseases caused by mitochondrial mutations are maternally inherited
What are the two the basic mechanisms of organellar genome inheritance?
1: Uniparental inheritance: 2: Biparental inheritance
What is the basic procedure of recombinant DNA technology
1: generating specific DNA fragments using restriction enzymes 2: joining these fragments with a vector 3: transferring the recombinant DNA molecule to a host cell to produce many copies that can be recovered from the host cell.
Understand the procedure for creating Recombinant DNA with restriction enzymes. (Slide 10-11)
1: produces DNA fragments with cohesive ends (sticky ends) 2: ssDNA fragments from different sources can anneal (stick together) by hydrogen bonding 3: DNA fragments will seal phosphodiester backbone 4: joins restriction fragments covalently to produce intact DNA molecules
How is transformation achieved?
1: using calcium ions and brief heat shocl to pulse DNA into cells 2: Electroporation: which uses a brief but high-intensity pulse of electricity to move DNA into bacterial cells
Mass spectrometry uses what ratio of different ions in sample?**
A mass-to-charge (m/z) ratio
Know the definition recombinant DNA technology
A set of techniques for amplifying, maintaining, and manipulating DNA sequences in vitro and in vivo.
Gene editing with CRISPR-Cas9 has been used to delete mutant exon 23 from a mouse model ofDuchenne Muscular Dystrophy (DMD) loss of function alleles in _________ gene
Dystrophin
restriction site and type fragment produced for HindIII
A/AGCTT, sticky end
What was the benefit of whole-genome sequencing?
All DNA fragments in a genomic sample are sequenced without the need to insert DNA fragments into vectors and cloning them in host cells
What is a plasmid?
An extrachromosomal, circular DNA molecule that replicates independently of the host chromosome
Color of final mice and gene expression. Brown v. black (+/+, +/-, -/-)
Brown
what is genomic imprinting controlled by?
By imprinting centers (IC) located nearby the imprinted areas in same chromosome
What is the best approach for genome editing?
CRISPS-cas
True/False: germinal gene therapy has been performed in humans.
False
True/False: PacBio SMRT sequencing uses a nanopore and electrical signature for each nucleotide
False it passes through a polymerase in an illuminated volume then uses nucleotide incorporation with flourescent signalSZ
True/False: Next-generation sequencing uses in vivo cloning and amplification?
False it uses in vitro and polony generation. Old sanger sequening uses in vivo and amplification
Genomic libraries contain all of the ______
DNA
What are the differences between DNA, genomic, and cDNA libraries?
DNA library: collection of cloned fragments of DNA, usually from a single source Genomic library: libraries derived from the genomic DNA of an organism cDNA(Complementary DNA): those derived from mRNA isolated from cultured cells or tissues
Cloning vector
DNA molecules that accept DNA fragments
True/False: CRISPR-Cas9 uses sgRNA and dsDNA break repair mechanisms.
False, they are used to either disrupt gene function or create nonfunctional allele
reaction site and type fragment produced EcoRI
G/AATTC, sticky end
restriction site and type fragment produced for BamHI
G/GATCC, sticky end
What are orthologs?
Genes from different species thought to have descended from common ancestor
What is the most important part of building a genome sequence?
Genome annotation
What are the active histones?
H3K4me3 H3K9Ac H3K36me3
What are the repressive histones? (slide 25)
H3K9me3 - histone H3 - lysine 9 - trimethylation H2K27me3(inactive X)
What are the differences between homoplasmy/heteroplasmy?
Homoplasmy: contain organelles with the same genotype; a cell or organism in which all copies of an organelle gene are the same Heteroplasmy: a cell or organism in which not all copies of an organelle gene are the same; cells contain a mixture of alleles
What is the maternal effect and how does the maternal genes affect the phenotype of the offspring?
Implies that an offspring's phenotype is under the control of nuclear gene products present in the egg. The genotype of the female parent and not that if the embryo determines the phenotype of the offspring.
What is organellar inheritance?
Refers to transmission of genes on mitochondrial and chloroplast chromosomes, as opposed to nuclear chromosomes.
What is a clone?
The recovered copies of a recombinant DNA molecule, and can be used to study the structure and orientation of the DNA
What is the main difference between mendelian and non-mendelian inheritance?
Mendelian - Each parent contributes an allele to their offspring - If the genotypes of the parents are knows, Mendel's 4 postulates can be used to estimate the genotypes and phenotypes of the offspring Non-Mendelian - Principles of Mendelian inheritance apply, but the manisfestation of the trait does not
How do restriction enzymes help with WGS and assembly? Know the examples discussed in class and how many contigs each one can give (Slide 14)
The restriction enzymes are used to digest genomic DNA into contigs, which then can be sequences and aligned using bioinformatics to identify oevrlapping fragments based on sequence identity - EcoRI digestion produces two fragments: contigs 1 and 2-4 - BamHI produces three fragments: Contigs 1-2,3, and 4
What is genomics (Genomic Analysis)?
The study of whole genomes or genomes in their entirety - the most repidly advancing area of modern genetics - provides unprecedented information about genomes of different organisma
What is a contig and know the process of alignment of them to make a scaffold
They are continuous fragments that use overlapping fragments which are adjoining segments that collectively form one continuous DNA molecule within the chromosome
T/F: WES fails to identify gene regulatory regions that influence gene expression?
True
True/False: Both subunits of insulin are needed to unite and make active insulin?
True
True/False: Insulin was among the first human genes to be expressed in E. coli.
True
True/False: Next-generation sequencing uses cyclic flowcell sequencing?
True
True/False: Transgenic coho salmon express the growth hormone gene only in spring and summer?
True
True/False: mtDNA is smaller than DNA in chloroplasts?
True
Proteomics uses ___________ for separating hundreds of thousands of ________ with high resolution.***
Two-dimensional gel electrophoresis (2DGE): proteins
What is totipotent?
Under the appropriate condiions a normal plant can be regenerated from a single or isolated plant cell
The closest living relatives of mitochondria are free-living ________?
alpha-proteobacteria
Know the difference between a sticky and blunt end.
Sticky ends: single stranded segments produced by some restriction enzyme cuts; can base pir with complementary sequences Blunt end: no single stranded overhangs
Restriction enzymes bind to what?
binds to to DNA at a specific recognition sequence(restriction site)
restrication site and type fragment produced for AluI
blunt end
EcoRI, and AluI and what fragment is produced (Slide 9)
blunt/sticky
What is the insert size for a BAC(Bacterial artificial chromosomes)?
capacity of 100-200 kb
What is a plastid?
chloroplasts are the most familiar of various organelles derived from a precursor organelle
What is subcloning?
cutting of DNA into smaller fragments in order to separately clone subsections of the sequence.
The closest relatives of chloroplasts are free-living ________?
cyanobacteria
When are eukaryotic expression vectors used?
in yeast or tissue culture cells in situations where bacteria cannot produce functional products of a transgene
What is the percent of the genome that codes for proteins? (how many genes does that make***)
less than 2% genome - which is about 20000 genes
Alternative splicing Human Genome Project revealed the number of genes is ______ than the number of predicted genes
lower
Majority of genes needed for proper mitochondrial function are located in the _____?**
nucleus(>1000)
What is transformation?
taking up naked exogenous (outside) DNA
The steps of whole-genome sequencing
1. Genomic DNA cut with restriction enzymes to create series of overlapping fragments 2: Overlapping fragments aligned using computer programs to assemble entire chromosome 3: Fragments are alined based on identical DNA sequences - creates contig
What is a BAC/YAC
BAC = Bacterial artificial chromosomes: the preferred artificial chromosome for use as a cloning vector, generally very large bt low copy number plasmids YAC = yeast artificial chromosomes: only carry DNA and do not signal for protein
Genetically modified bacterial plasmids - first vectors developed**
Bacterial Plasmid Vectors
What transgenic model system did we use as an example in class and what was it trying to achieve? (slides44-46)
Creating a Loss-of-Function CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) Allele in Mice Through Homologous Recombination
Knockout mice used ________ cells to be injected into a blastocyst.
Embryonic stem (ES)
What is genomic imprinting?
Epigenetic phenomenon resulting in the differential alleles expression of certain genes. This leads to the expression of one allele and repression of the other allele. It is rare, naturally occurring and occurs in placental mammals and flowering plants.
What are Contigs-continuous fragments?
It is a part of the bioinformatics applications and is where overlapping fragments adjoin segments that ollectively form one continous DNA moleculs within the chromosome.
What is bioinformatics used for and algorithms(go to slide 15,23-24 for last part of question)?
It is used to provide unprecedented information about genomes of different organisms such as their gene structure, sequence and expression, and protein structure and function. The algorithim-based software programs create DNA sequence alignment. Meaning similar sequences of bases are lined up for comparison. This identifies overlapping sequences and allows scientists to reconstruct their order in the chromosome
What is the symmetry that recognition sequences exhibit and how long can they be?
Palindrome, can have mostly four to six nucleotides, some contain eoght or more
What are the other types of vectors?
Phage vectors, Bacterical artificial chromosomes, yeast artificial chromosomes
Difference between RT-PCR and qPCR.
RT-PCR(Reverse transcription PCR): methodologt for studying gene expression (mRNA production by cells or tissues) qPCR(Quantatice real-time PCR): Real-time PCR allows researchers to quantify amplification reactions as they occur in real time
How can selectable markers identify recombinant DNA and what are examples of the genes that are used?
They ditsinguish hot cells that have taken them up from those that have not. Genes conferring resistance to antibiotics are commonly used.
___________ and soil bacterium can be used for introducing genes in plants.
Ti plasmid
What is used for resequencing home genomes?
reference-based genome assembly
What is endosymbiont theory?
posits that eukaryote arrival corresponded to larger prokaryotic cells engulfing smaller, free-living prokaryotes that became mitochondria and chloroplasts. Mitochondria and chloroplasts arose independently about 2 billion years ago from free-living bacteria tat possessed the abilities now attributed to these organelles - aerobic respiration and photosynthesis.
Transcriptome analysis can study expression of genes by the genome ____________ and___________.
qualitatively; quantitatively
What is a genome?
the haploid set of chromosomes in a gamete or the complete set of genes or enetic material prsent in a cell of an organism
