ch 19 genetics
replication by PCR requires a ___ _____ from which a new DNA strand can be copied, and a pair of single stranded primers, each with a ___ group to which new nucleotides can be added. the primers used in PCR are short fragments of DNA, typically 17-25 nucelotides long, that are ______ to known sequences on the template. To carry out PCR, researcher begin with a solution that includes the target DNA, DNA polymerase, _______, primers, and magnesium ions and other salts that are necessary for the reaction to proceed
3'OH; complementary; dNTP (four deoxyribonuceloside triphosphates/ the substrates for DNA polymerase)
the sequences recognized by restriction enzymes are usually from ___ to ____ bp long. most recognition sequences are ______--sequences that read the same (5' to 3') on the two complementary DNA strands
4;8; palindromic
is the typing of an individual for genetic markers at highly variable loci. this is useful for forensic investigators, to determine whether the suspect could have contributed to the evidentiary DNA obtained from blood or other bodily fluids found at the scene of a crime. other applications include paternity testing and the identification of bodily remains.
DNA fingerprinting
a collection of clones containing all the DNA fragments from one source is called a _____ ______. for example, we might isolate genomic DNA from human cells, break it into fragments, insert the fragments into vectors, and clone them in bacterial cells. the set of bacterial colonies or phages containing these fragments is human ______ ________, containing all the DNA sequences found in the human genome
DNA library; genomic library
DNA fragments with sticky ends can be "glued" together: any two such fragments cleaved by the same enzyme can be joined together permanently by ____ ____, which seals nicks between the sugar phosphate groups of the fragments
DNA ligase
determines the sequence of bases in a DNA molecule. sequencing allows the genetic information in DNA to be read, providing an enormous amount of information about gene structure and function
DNA sequencing
PCR can be used to detect the presence of a particular DNA sequence in a sample. modern diagnostic test for this infection with HIV, the causative agent of AIDS, use this of PCR amplification of HIV sequences. identify genetic variation in natural populations , identify diffrences in nucelotide sequences primers specific to a particular DNA variant can be used to determine whether that variant is present in the genome of an individual organism. allowed the isolation of DNA from ancient sources, crime sites used to introduce new sequences into a fragement of DNA. primers can be designed to contain new restriction sites or other desirable sequences at the 5' ends. these sequences will be copied by PCR and thus added to the ends of the amplified DNA modification of PCR, real time PCR, can be used t measure the starting amount of nucelic acd
PCR uses
labeled probed, which are sequences of ____ or _____ are complementary to the sequence of interest, can be used to locate ____ ______ or ______ among DNA fragments separated by electrophoresis
RNA; DNA; individual genes; sequences
____ _____ restriction enzymes recognize specific sequences and cut the DNA to defined sites within or near the recognition sequence. virtually all molecular genetics is done with this
Type II
what happens in a typical restriction reaction?
a concentrated solution of purified DNA is placed in a small tube with a buffer solution and a small amount of restriction enzyme. the reaction mixture is then heated at the optimal temperature for the enzyme(37C). often within an hour, the enzyme cuts the appropriate restriction sites in all the DNA molecules
what are the 3 important characteristics of an effective cloning vector
an origin of DNA replication so they can be maintained in a cell. a gene, such as antibiotic resistance, to select for cells that carry the vector. a unique restriction site or series of sites to where a foreign DNA molecule may be inserted
there some restriction enzymes that cuts in the middle of its recognition sequence and cuts on the two strands are directly opposite of each other, producing ____ _____ fragments that must be joined together in other ways
blunt ends
an alternative to creating a genomic library is to create a librabry consisting only of those DNA sequences that are transcribed into mRNA called ______ ______ because all the DNA in this complementary to mRNA.
cDNA
1) it is enriched with fragments from actively transcribed genes 2) introns do not interrupt the cloned sequences; introns would pose a problem when the goal is to produce a eukaryotic protein in bacteria because most bacteria have no means of removing the introns
cDNA library advantages
in dideoxy sequencing, what terminates DNA synthesis?
ddNTPs because they lack a 3'OH group
PCR: first, the double stranded template DNA is ________ by high temperature. then ______ corresponding to the ends of the DNA sequence to be amplified are ______ to the single stranded DNA template strands. these primers are extended by a thermostable DNA polymerase so that the target DNA sequence is duplicated. these steps are repeated 30 times or more. each cycle of _____, ______ _______, and _______ results in the doubling the number of copies of the target sequence between the primers
denatured; primers; annealed;denaturation; primer annealing; extension
Sanger created the ______ _______ method, which is based on the elongation of DNA by DNA polymerase
dideoxy sequencing
method of DNA sequencing is based on replication. the fragment to be sequenced is used as a template to make a series of new DNA molecules. in the process, replication is sometimes, but not always terminated when a specific base encountered, producing DNA strands of differnet lengths, each which ends in a known base.
dideoxy sequencing
method relies on the use of special substrate for DNA synthesis. normally, DNA is synthesized from dNTPs, which have aOH group on the 3' carbon atom. this method uses special nucelotides, ddNTPs that are also used as substrates. the ddNTPs are identical with dNTPs, except that they lack a 3'OH group. in the course of DNA synthesis, ddNTPs are incroporated into a growing DNA strand. however, after a ddNTP has been incorporated into the DNA strand, no more nucelotides can be added because there is no 3'OH group to form a phosphodiester bond with an incoming nucelotide. thus, ddNTPs terminate DNA synthesis
dideoxy sequencing
PCR: each cycle of denaturation, primer annealing, and extension results in...
doubling the number of copies of the target sequence between the primers
in addition to the usual origin of replication, restriction sites, and selectable markers, contains sequences required for transcription and translation inn bacterial cells
expression vector
after DNA fragments have been separated by gel electrophoresis, DNA molecules(nucleic acid) can be visualized staining with a ______ dye, such as ethidium bromide, which ______ between the stacked bases of the DNA double helix, and the dye-DNA complex when irradiated with an ___ light source. alternatively, they can be visualized by attaching _____ or ______ labels to the DNA before it is placed on the gel
fluorescent; intercalates; UV; radioactive; chemical
an approach which begins with a phenotype (a mutant individual) and proceeds to a gene that encodes the phenotype
forward genetics
to use a probe; a researcher first cuts the DNA into ________ by using one or more restriction enzymes and then separates the fragments with _____ ________. next, the separated fragments must be denatured and transferred to a permanent solid medium(such as nitrocellulaose or nylon membrane). _____ _____ is one technique used to transfer the denatured (single stranded) fragments from a gel to a permanent solid medium. after the single standed DNA fragemnt have been transferred, the membrane is placed in ______ solution containing a labeled probe. the probe binds to only a part of a DNA fragment, so the DNA fragment may contain sequences not found in the probe. the membrane is then washed to remove any unbound probe; a biochemical method reveals the presence of the bound probe. RNA can be transferred from a gel to a solid support by a related procedure called ____ ____. hybridization with a probe can reveal the size of a particular mRNA molecule, its relative abundance, or the tissues in which it is transcribed. _______ _______ is the transfer of protein from a gel to membrane. here, the probe is usually an antibody, usued to determine the size of a particular protein and the parttern of te protein's expression
fragments; gel electrophoresis; southern blotting; hybridization; northern blotting; western blotting
restriction enzymes cut foreign DNA, such as viral DNA, into _______. Bacteria protect their own DNA by modifying bases, usually by _______, at the _______ ________
fragments; methylation; recognition sites
PCR steps. 1) a starting solution of DNA is heated to 90-100C to break the ____ _____ between the strands and thus produce the necessary single stranded templates. the reaction mixture is heald at this temperature for a minute or less. 2) the DNA solution is cooled quickly to 30-65 C and heald at this temperature for a minute or less. during this short interval, the DNA strands do not a chance to ________, but the primers are able to attach to template stands. 3) the solution is heated for a minute or less to 72C, the temperature at which _____ ______ can synthesize new DNA strands. 'at the end of the cycle, two new double stranded DNA molecules are produced for each original DNA
hydrogen bonds; reanneal; DNA polymerase
involves hybridization of radiolabeled or fluorescently labeled DNA or RNA probes to DNA or RNA molecules that are still in the cell. this technique can be used to visualize the expression of specific mRNAs in different cells and tissues and location of genes on metaphase or polytene chromosomes
in situ hybridization
what are the steps of gene analysis?
isolate, recombine, then amplify
western blotting is the transfer of a protein from a gel to membrane, why id the probe usually an antibody>
it is used to determine the size of a particular protein and the pattern of the protein's expression
_____ _____ have a target gene disrupted or deleted. first, the target gene is cloned. the middle portion of the gene is replaced with a selectable marker, typically the neo gene that cionfers resistance to the drug G418. this construct is then introduced back into mouse embryonic stem cells and cells with G418 resistance are selected. the surviving cells are screened for cells where the chromosomal copy of the target gene has been replaced with the neo containing construct by homologous recombination of the flanking sequences. these embryonic stem cells are then injected into mouse blastocyst-stage embryos and these chimeric embryos are transferred to the uterus of a pseudopregnant female mouse. the ____ cells will participate in the formation of many tissues in the mouse fetus, including germ line cells. the chimeric offspring are interbred to produce offspring that are homozygous for the _______ allele. the phenotypes of the ______ mice provide information about the function of the gene.
knockout mice
there is a relationship between the ____ of the recognition sequence and the ____ ___ ______ it is resent in a genome
length; number of times
restriction enzymes that recognize longer sequences will....than will restriction enzymes that recognize sequences
longer sequences will cut a given piece of DNA into fewer and longer fragments
restriction enzymes recognize specific nucleotide sequences in DNA and make double stranded cuts at those sequences. are produced naturally by bacteria and used in defense against viruses. a bacterium protects its own DNA from restriction enzymes by modifying the recognition sequence, usually by adding ______ _____ to its DNA
methyl groups
gel electrophoresis uses an electric field to drive DNA molecules through a get that acts as a ______ _____. the gel is an aqueous matrix of ______ or _______. DNA molecules are loaded into a slot or well at one end of the gel. when an electric field is applied, the negatively charged DNA molecules migrate toward the positive electrode. Shorter DNA molecules are ____ hindered by the aqueous matrix and migrate faster than longer DNA molecules, which must wind their way around obstacles and through the pores in the gel matrix
molecular sieve; agarose; polyacrylamide; less
RNA can be transferred from a gel to a solid support. hybridization by a probe can reveal the size of a particular mRNA molecule, its relative abundance, or the tissues in which it is transcribed
northern blotting
in _______ _____ _____, an oligonucelotide containing the desired mutation in the sequence is synthesized. this mutant oligonucleotide is annealed to denatured target DNA template and used to direct DNA synthesis. the result is a double stranded DNA molecule with a mismatch at the site to be mutated. when transformed into bacterial cells, bacterial repair enzymes will convert the molecule to the mutant form about 50% of the time.
oligonucleotide directed mutagenesis
what are the common vectors?
plasmids, cosmids, BACs, YACs
enzyme that recognizes particular base sequences in DNA and makes double-stranded cuts nearby
restriction enzyme
variations in DNA sequence that can be detected by cutting the DNA with restriction enzymes. genetic markers useful in mapping
restriction fragment length polymorphisms
approach beginning with a genotype--a DNA sequence--and proceed to the phenotype by altering the sequence or inhibiting its expression. a geneticist might begin with a gene of unknown function, induce mutations in it, and then observe the effect of these mutations on the phenotype of the organism
reverse genetics
once a DNA library is created, it can be screened to find a gene or sequence of interest. the screening procedure used depends on what is known about the gene. they use probes , or use a similar gene from another organism as a probe. successful hybridization does not require perfect complementarity between the probe and the target sequence, so the related sequence can often be used as the probe. synthetic proes can be created if the protein produced by the gene of interest has been iolated and its amino acid sequence has been determined loof for the protein product of a gene. requires that the DNA library be cloned in an expression vector
screening DNA libraries
Gel electrophoresis can be used to separate DNA molecules on the basis of their ____ and ______
size; electrical charge
technique used to transfer the denatured (single-stranded)fragments from a gel to a permanent solid medium
southern blotting
some restriction enzymes make ____ ____ in the DNA, for example Hind III cuts the sugar phosphate backbone of each strand generating fragments with short, single stranded overhanding ends. such ends are _____ ______, because they are complementary to each other and can spontaneously pair to connect fragments.
staggered cuts; cohesive ends (sticky end)
the first loci used for DNA fingerprinting were variable number of ____ _____ (VNTR) loci; these consist of short tandem repeat sequences located in introns or spacer regions between genes. the number of repeat sequences at the locus does not affect the _______ of the individual in a discernable way, so the number of repeats at each of these loci are highly variable in the population. more recently, loci with smaller repeat sequences of just a few nucelotides, called _______ _______ _______ (STRs), have been adopted because they can be amplified by PCR. the variable number of repeats creates PCR fragments of different sizes. genotyping at 13 to 15 of these unlinked STR loci can identify one individual amoung trillions of potential genotypes.
tandem repeat; phenotype; short tandem repeats
hybridization with a prove can reveal....
the size of a particular mRNA molecule, its relative abundance, or the tissues in which it is transcribed
an organism that has been permanently altered by the addition of a DNA sequence to its genome is said to be transgenic, and the foregin DNA that it carries is called a ________
transgene
is the transfer of a protein from a gel to membrane. here the probe is usually antibody, used to determine the size of a particular protein and the patter of the protein's expression
western blotting
when are restriction enzymes used?
whenever DNA fragments must be cut or joined