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reverse transcriptase

enzyme that catalyzes synthesis of cDNA from RNA template

restriction endonuclease

enzyme that cuts DNA at specific sequences (palindromic sequence)

DNA ligase

enzyme that catalyzes the formation of a phosphodiester bond between nucleotides

plasmids

small, extrachromosomal loops of DNA found in many bacteria

Taq polymerase

DNA polymerase used in PCR (annealing, adds the primers and bases to cDNA)

SNP

single nucleotide polymorphism, site in DNA where the identity of the base varies among individuals in a population

recombinant DNA technology

taking a copy of a gene from one individual and placing it in a genome of different individual.

genetic libraries

collection of all DNA sequences present in a particular source. The library consists of individual DNA fragments that are isolated and inserted into a plasmid/vector

DNA probing

use of labeling known DNA fragments to find a particular DNA sequence in a large collection

PCR

polymerase chain reaction, DNA synthesis reaction that uses known primer sequences on either side (opposite)

dideoxy sequencing

ddNTPS, to determine base sequence of a gene or other sections of DNA

genetic mapping

creation of a map showing the relative positions of genes or specific DNA sequences on chromsomes. Analyzing the frequency of recombination between sequences

apoptosis

when CAG from huntington's disease builds up in the brain, triggers neurons to program cell death

transgentic

alleles that have been modified by genetic engineering

prototrophs

able to synthesis all organic material, can grow in minimal medium

auxotrophs

a mutation that organism loses the ability to synthesis organic material

genetic recombination (bacteria)

replacement of 1 or more genes present in 1 strain with those from a genetically distinct strain

cotransformation

genes very close together to another to make a single segment (linked genes)

heteroduplex

host and mutant cell

Name of bacteria causing viruses

firmicutes

Bacteria linked to nervous system, GI tract, causes food poisoning

clostridium botulinum

Bacteria linked to wounds, nervous system, causes tetanus

clostridium tetani

Bacteria linked to skin, urogenital canal, causes acne, boils, impetigo, toxic shock syndrome

staphylococcus aureus

Bacteria linked to respiratory tract, causes pneumonia

streptococcus pneumoniae

Bacteria linked to respiratory tract, causes strep thorat, scarlet fever

streptococcus pyogenes

vector

plasmid used to make copies of a foreign DNA sequence

inserting genes into a plasmid?

identify palindromic recognition site, add restrictions endonuclease to make cuts at sites, sticky ends, insert gene into plasmid, DNA ligase catalyze formation of phosphodiester bond

sticky ends

restriction enzymes cut covalent phophodiester bond of both strands often in a staggered way creating single stranded ends, sticky ends. these extensions will from hydrogen bonded base pairs with complementary single stranded stenches on the other DNA molecule

cDNA library

DNA versions of all the mRNAs in a particular cell

transduction

DNA is transferred from one bacterium by another by a virus (gene therapy)

flow chart of bacteria to recombinant DNA

bacteria (structure, plasmid) --> bacterial genetics (transformation, conjugation, transduction) --> recombinant DNA (tech)

BAC

bacterial artificial chromosomes (used for shotgunning), able to replicate large segments of DNA

BAC library

a set of all DNA sequences in a particular genome, split into small segments and inserted into cloning vectors

shotgun sequencing

genome sequence is cut into 160kb fragments, cloned into BAC, sonicated into 1 kb, cloning using plasmids, sequence each fragment, assemble the fragments through overlapping.

difference between pyrosequencing vs. dideoxy sequencing

pyrosequencing happens on a single DNA fragment rather than many copies of the same fragment for dideoxy sequencing

Benefits of pyrosequencing

miniaturization that makes it possible to sequence many DNA fragments at the same time and avoiding slow and expensive steps like cloning fragments into BACs and plasmids to produce many copies of each fragment

bioinformatics

field that fuses computer science and biology in an effort to manage, analyze, and interpret biological information.

open reading frames

sequences typical of promoters, operators, or other regulatory sites. Represent candidate genes

homologous genes

related by descent from a common ancestor

EST

expressed sequence tag, representing protein-coding genes. Used to find the matching sequence in genomic DNA

Most productive strategy to finding a gene in euks

isolate mRNAs from cells, use reverse transcriptase to produce a cDNA version of each mRNA, and sequence a portion of the resulting molecule to produce an EST.

lateral gene transfer

the movement of DNA from one species to another. Extenstive in prokaryotes

2 general criteria for lateral transfer

gene is much more similar to genes in distantly related species than to those in closely related species AND proportion of G-C base pairs to A-T base pairs in a particular gene or series of genes is markedly different from the base composition of the rest of the genome

environmental sequencing/metagenomics

study of genes (not organisms). Start by studying a simple system, then go on to more complex situations. It catalogs all the genes from in a particular habitat

True or false: In bacteria, there is a general correlation between the size of the genome and the metabolic capabilities of the organism

true

Why is it hard to sequence eukaryotic genes?

too big and many repeating DNA sequences that occur between genes or inside introns and do not code for products used by the organism

transposable elements

segments of DNA that are capable of being inserted into new locations, or transposing, in a genome. They are known to be selfish gene.

Why isn't a transposable element like a virus?

Because a virus leaves the host cell once they have infected it. Transposable elements makes copies of themselves and never leave.

LINE

a type of selfish gene (transposable element), long interspersed nuclear element.

How line transposable elements spread?

a LINE exists in DNA, RNA polymerase transcribes into LINE mRNA, LINE mRNA and protein enters the nucleus AGAIN, reverse transcriptase makes LINE cDNA and makes cDNA double stranded, integrase cuts chromosomal DNA and inserts LINE cDNA, new copy of LINE is integrated in genome

STRs

short tandem repeats, small sequences repeated one after another along a chromosome

two types of STRs

microsatellites (simple sequence repeats) and minisatellites (VNTRs: variable number tandem repeats)

VNTRs

variable number tandem repeats, 6-500 bases long, originated when DNA polymerase skips/mistakenly adds extra bases during replication

what does it mean that microsatellite and minisatellite loci are "hypervariable"?

they vary among individual much more than any other type of sequence does as a result of unequal crossover

unequal crossover

homologous pairs line up incorrectly during prophase of meiosis I, two chromosomes pair in a way that matches up bases with different DNA repeats, when crossover occurs, the resulting chromosomes will have different number of repeats

Why are microsatellite and minisatellite sequences used for DNA fingerprinting?

Because they vary so much among individuals, it is a unique feature of their genomes

How do you fingerprint an individual?

obtain DNA, PCR using primers that flank a region containing an STR, once region has been cloned, it can be analyzed to determine the number of repeats present.

gene family

genes that are extremely similar to each other in structure and function (within a species)

gene duplication

extra copy of a gene is added to the genome

most common type of gene duplication results in...?

unequal crossover during meiosis

pseudogene

member of a gene family that resembles a working gene but does not code for a functional product. Made by mutations in the duplicated region of a gene.

Why is it that humans have fewer than 20,000 genes but can produce 100,000 different transcripts?

alternative splicing hypothesis: multicellular eukaryotes do not need enormous numbers of distinct genes. Instead alternative splicing creates different proteins from the same gene.

If humans and chimps are 98.9% identical, why do they look and act so differently?

even though the structural genes are identical, regulatory sequences and regulatory genes are very different.

regulatory sequence

section of DNA involved in controlling the activity of other genes; it may be a promoter, an enhancer

regulatory genes

code for regulatory transcription factors that alter the expression of specific genes

structural gene

sequence that codes for a tRNA, rRNA, protein, or other type of product

What recent discovery made scientists realize the eukaryotic gene expression is much more complex than previously thought?

many miRNA (regulate lifespan of mRNAs) genes and TUFs (transcripts of unknown function that never leave the nucleus)

DNA microarray

consists of a large number of single-stranded DNA segments that are permanently affixed to a glass slide.

How to set up DNA microarray?

isolate mRNA and use reverse transcriptase to make cDNA, make cDNA probes (using fluorescent tags to mark each cDNA), probe a microarray (cDNA probes will bind to complementary DNA sequence on the slide), shine laser light on one spot to induce fluoresence

What does a microarray help scientists do?

helps them study the expression of thousands of genes at a time. As a result, they can identify which sets of genes are expressed in concert under specific sets of conditions.

transcriptome

complete set of genes that are transcribed in a particular cell

proteome

complete set of proteins that are produced

proteomics

large-scale study of all the proteins in a cell/organism

What evidence do biologist use to infer that a gene is part of a gene family?

its sequence, structure and composition are similar to those of another gene in the same genome

Ames test

number of different strains of the bacterium that have been selected for their ability to reveal the presence of specific types of mutations (some have been to detect various frameshift mutations and other strains to detect base-pair subs

What is the reason for an Ames test?

to estimate mutagenicity and cancer-causing potential of chemical agents by following the rate of mutation in specific strains of bacteria

carcinogens

cancer-causing agents from the transformation of cells to the malignant state occurs as a result of mutations

What do bacteria have in common with archaea but not with eukarya?

absence of nucleus

How did bacteria change Earth's atmosphere to enable aerobic respiration?

oxygenic photosynthesis by photosynthetic bacteria significantly increased the amount of oxygen in the atmosphere.

Bacteria normally contain their genome in

nucleiod region

Plasmids are best thought of as

auxiliary genetic elements

Flagella and pili are made of

protein

Name the general category into which double-stranded circular extrachromosomal DNA elements such as F factors, ColE1, and R would fall.

plasmid

Hfr bacteria function as ____ in crosses with ___ bacteria and are called so because they result in ____ recombination.

donors, F-, high frequency

Why is it easier to identify spontaneous mutations in bacteria than in most eukaryotes?

They are expressed directly in descendant cells because bacteria are
haploid.

In an Hfr mating with an F- cell, why doesn't the recipient cell always become an Hfr or F+ cell?

The F factor is the last part of the chromosome to be transferred

True or false: An E. coli nutritional mutant is described as an auxotroph, which requires minimal medium with supplementation of an organic carbon source or inorganic ion for growth

true

What limits bacterial growth within a culture of medium?

nutrients and oxygen

Many identical copies of genes cloned in bacteria are produced as a result of _____.

plasmid and bacterial replication

What is the characterization of cutting a sequence by a restriction enzyme?

the sequence is usually palindromic

Imagine that you've isolated the complete human growth hormone gene directly from the human genome. After running through all the steps described in Chapter 19 for
cloning and gene expression in bacteria, you find that no human growth hormone is expressed. What is the most likely explanation?

bacteria cannot carry out splicing

If mRNAs could be ligated and replicated within plasmids, what enzyme commonly used in recombinant DNA technology would no longer be needed?

reverse transcriptase

Which of the following is a correct difference between a gene library and a gene clone?

A gene library contains many different cloned DNA sequences; a gene clone contains one type of DNA sequence

If a scientist needs to put new DNA into a bacteria, she must _____ the bacteria.

transform

A gene library from a particular human's retinal cell would be?

a collection of different DNA fragments ligated into plasmids

When constructing a cDNA library from a particular organism, the choice of tissue is _____, whereas when constructing a genomic DNA library from the same organism, the choice of tissue is _____.

critical, immaterial

How can an amino acid sequence be used to design a gene-specific hybridization probe?

All possible nucleotide sequences that could encode a portion of the polypeptide can be synthesized and used as probes.

Imagine that you've prepared a Southern blot and probed it using a single probe. You observe eight different bands after autoradiography, with one band being very dark and
the other seven being significantly lighter. What is suggested by these results?

The DNA did not transfer from the gel to the nylon filter.

What is a primary difference between PCR and traditional cloning procedures such as those used to clone the human growth hormone gene?

PCR eliminates the need for restriction enzymes, vectors, and cells.

Most important information for the success of PCR?

finding/using the right primers

In early forensic applications of DNA fingerprinting, DNA was extracted from crime scene material, digested with restriction enzymes, and then used to prepare a Southern blot. Today, PCR is used in the early steps of forensic DNA analysis. What advantage does PCR provide over the former method?

PCR requires much less DNA for analysis.

In a single PCR cycle consisting of 15 seconds at 94°C, 30 seconds at 50°C, and 1 min at 72°C, what is happening in the step run at 50°C?

Primers are annealing to the DNA to be amplified.

Why is it essential that genetic markers used in mapping disease genes be polymorphic?

If the marker isn't polymorphic, then it's impossible to use genetic mapping techniques to establish an association between the marker and the disease gene.

Imagine that you compare two DNA sequences found in the same location on homologous chromosomes. On one of the homologs, the sequence is AACTACGA. On the other homolog, the sequence is AACTTCGA. Within a population, you discover that each of these sequences is common. These sequences _____.

contain an SNP that may be useful for genetic mapping

An early step in shotgun sequencing is to _____

break genomic DNA at random sites

The bulk of the sequence data in whole genome sequencing comes from _____.

relatively small (~ 1000 base-pair) sequences cloned into plasmids

A scientist needs to find a collaborating scientist to help her with organizing a large amount of sequence data from the genome of a previously unsequenced and rare plant. This scientist should find a collaborator who specializes in _____,

bioinformatics

Biologists now routinely test for homology among potentially related species using DNA sequences. These tests reveal if the sequences from each species _____

are related by descent from a common ancestor

If the sequence of a cDNA matches a DNA sequence in the genome, then this genomic DNA is likely to _____.

code for a protein

In comparing DNA sequences from two different eukaryotic organisms, which do you expect to vary the most?

sequence that codes for introns

Scientists have begun discussing the possibility of putting an entire human genome (including that arising from endosymbiosis) into a cow egg cell without a genome. The
next generation of cells from the cow egg would have _____.

human genome only

In what sense are studies by 19th-century naturalists and those by early 21st-century genomic biologists similar?

Both focus on observing and describing what exists in their realms of investigation.

Even though bacteria are more simple than eukaryotes, genomic data on bacteria was surprising because _____.

there are many genes of unknown function

Imagine that you've sequenced the genome of a human pathogenic bacterium. In the early stages of analysis, you discover a stretch of DNA that has a significantly different GC content (the proportion of bases that are G and C). Further examination of this region shows there are
roughly one dozen protein-coding regions. These are not found in the genome of a previously sequenced and related bacterium. These sequences do, however, predict protein products strikingly similar to those of another bacterial pathogen that is not closely related to the organism
you're studying. You immediately suspect _____.

lateral gene transfer

Why are transposable elements considered to be selfish genes?

They replicate using the host's resources without direct benefit to the host

The mechanism for the spread of LINES involves some unusual features, including _____.

the transport of mRNA from the cytoplasm to the nucleus

Although transposable elements and simple tandem repeats (STRs) are both repetitive DNAs, they differ in that _____.

the repeated unit in STRs is clustered one after another; transposable element repeats are scattered throughout the genome

Forensic DNA fingerprinting often involves recovery of minute quantities of DNA from a crime scene. What is a concern in analyzing this DNA?

It is critical to avoid the introduction of contaminating human DNA unrelated to the crime

In eukaryotes, the major mechanism of introducing new genes into the genome is _____.

duplication and divergence

If alternative splicing did not occur, a prediction would be that _____.

the human genome would contain many more genes

A current view of how the human and chimpanzee can share most of their nucleotide sequence yet exhibit significant phenotypic differences is that many of the most important
sequence differences alter _____.

regulatory sequences and proteins

Like DNA microarrays, Northern blots analyze the levels of mRNA. However, Northern blots are almost always prepared with a probe tagged with one label, whereas DNA microarrays almost always use two groups of probes, each one tagged with a different fluorescent label. What
advantage is conferred by using two labels for the microarray probes?

The two labels allow the analysis of the expression of more genes.

RFLP

(Restriction Fragment Length Polymorphism) Differences in DNA sequence on homologous chromosomes that can result in different patterns of restriction fragment lengths (DNA segments resulting from treatment with restriction enzymes); useful as genetic markers for making linkage maps.

If the RFLP morph is always present when the disease allele is present, does that mean that the RFLP morph causes disease?

no, means they are closely linked

What is a strategy for identifying a eukaryotic gene?

is to use a cDNA library to identify mRNA sequences and then locate the gene in the genome sequence.

What are some different cloning vectors?

plamids, bacteriophages, plasmid vectors, cosmids, BAC, YAC

cosmids

viral DNA that has been modified as both a plasmid and virus

colony filter hybridization

transferring bacteria from a petri plate to a nitrocellulose filter. The colonies on the filter are then lysed, and the liberated DNA is fixed to the filter by raising the temperature to 80°C. After hybridization with a labeled probe, the position of the colonies containing the sequence under study is determined by autoradiography

General steps for molecular searching

separate molecules by size, transfer to solid support, prepare molecules for probing, detect where probe is bound

southern blotting

A technique that enables specific nucleotide sequences to be detected in a sample of DNA. It involves gel electrophoresis of DNA molecules and their transfer to a membrane (blotting), followed by nucleic acid hybridization with a labeled probe.

Steps of southern blotting

chromsomal DNA - cut DNA fragment to be detected, total cut DNA is run through electro gel, blot probed with cloned gene
*must be single stranged*

Why is DNA treated with alkaline solution during electrophoresis in southern blotting?

to break apart the hydrogen bond and create single-stranded DNA

steps to northern blot

chromsomal DNA - mRNA to be detected, total mRNA is run through electro gel, blot probed with cloned gene

steps to western blot

chromosomal DNA - protein product is to be detected, total protein, blot probed with anitbody to protein

western blot

uses antibody to identify a target protein, either on a gel or inside bacteria

is RFLF morph always present with a specific allele?

Yes, they migrate together with the disease

How many reading frames does each sequence have?

3

Why is it hard to sequence eukaryotic genomes by shotgunning?

Because there is frequent overlapping

What does a microarray mean? Interpret the colors

a certain color means the inhibition of the other color, which genes are turned on and off?

what are the two components of R-plasmid?

the resistance transfer factor (RTF, encodes genetic information essential to transferring) and r-determinant (genes that confer resistence to antibiotics)

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