GENETICS EXAM 5

cDNA

(complementaryDNA)is made from mRNA so reflects what genes were expressed in the particular cells from which the mRNA was isolated

DNA Sequencing

--Depends on chain termination 1)PCR w/ dideoxynucleotide 2) Gel Electrophoresis w/ fluorescent colored tags

amniocentesis

A technique of prenatal diagnosis in which amniotic fluid, obtained by aspiration from a needle inserted into the uterus, is analyzed to detect certain genetic and congenital defects in the fetus. ---Geneticists PCR amplify the disease locus from genomic DNA prepared from these fetal cells and then analyze the PCR products by sequencing or sizing.

cDNA

After infecting a cell, a retrovirus uses reverse transcriptase to copy its single strand of RNA into a strand of complementary DNA or _________. -----Reverse transcriptase can also function as a DNA-dependent DNA polymerase, to make a second strand of DNA complementary to first cDNA strand (equivalent sequence to original RNA template).

small interfering RNAs siRNAs

Block translation/destabilize mRNAs ---recruit histone modifying enzymes to DNA, resulting in heterochromatin formation

Sanger vs Next gen sequencing

Both: DNA polymerase adds fluorescent nucleotides one by one onto a growing DNA template strand. Each incorporated nucleotide is identified by its fluorescent tag ---Main diff: Sanger method only sequences a single DNA fragment at a time, NGS is parallel, sequencing millions of fragments simultaneously per run

Chromatin immunoprecipitation-sequencing (ChIP-Seq)

Can be used to identify all the targets genes of a particular transcription factor --Point is to determine which DNA sequence a target protein binds to. 1)Covalently bind protein in cell to DNA, fragment DNA, then purify protein & DNA complex. To identify DNA sequence of fragments, protein needs to be removed.

DNA microarray

Contains allele-specific oligonucleotides (ASOs) for millions of SNP loci. Under the proper conditions, a probe made of fluorescently-labeled genomic DNA fragments binds only to complementary ASOs, allowing these loci to be genotyped.

protein domains

Exons often encode discrete________, each of which is a linear se- quence of amino acids that folds up in three-dimensional space so as to act as a single functional unit.

Simple Sequence Repeat

Polymorphism resulting from a tandemly repeated short DNA sequence

processed pseudogene

Produced through reverse transcription of mRNAs (in which double-stranded cDNA copies of the mRNAs are inserted into the genome).

deletion- insertion polymorphisms (DIPs)

Short insertions or deletions of a single or a few base pairs --problems in DNA replication or recombination, and mistakes that occur when cells try to repair damage such as broken DNA strands

allele-specific oligonucleotides

Short, single-stranded fragments of DNA used as probes to identify alleles that differ by a single oligonucleotide ---short 20- to 40-base-long oligo- nucleotides that will hybridize under the right conditions to only one of the two alleles at a SNP locus.

Microarray

Silicon or glass sheet with thousands of DNA probes that can be used to identify which genes in a tissue are expressed ---shows which genes are being actively transcribed in a sample from a cell

Northern blot

Similar technique [to Southern], except that Northern blotting involves radioactive DNA probe binding to sample RNA .

SSR polymorphisms

Single sequence repeats usually caused by stuttering of DNA Poly

reverse transcriptase

To produce DNA clones from mRNA sequences, researchers rely on RNA-dependent DNA polymerase, or__________ used by retroviruses

cDNA sequence

_____________ reveal how a primary transcript is spliced in a given cell type, and thus predict the amino acid sequence(s) of a gene's protein product(s) in that cell type.

reverse transcriptase

a polymerase that catalyzes the formation of complementary DNA (cDNA: sinlge strand) using RNA as a template ----then add dNA poly to make double strand DNA ---insert this DNA into cloning vector, amplify & sequence & put into cDNA library

RefSeq

a single, annotated version of a species' genome stored in GenBank to which the sequences of other genomes may be compared

BLAST

a software tool that allows scientists to find DNA or protein sequences that are similar to each other.

RNA Seq

a technology that uses the capabilities of next-generation sequencing to reveal a snapshot of RNA presence and quantity from a genome at a given moment in time --using High-throughput sequencing to coordinately examine all RNA transcripts in the cell at once

proteome

all the proteins made in an organism

paralogous genes

arise by duplication; this term is usually used to denote the different members of a gene family

micro RNAs miRNAs

bind to complementary RNA to prevent translation -block mRNA translation -destabilize mRNAs

homology

blanket term for all evolutionarily related sequences

syntenic blocks

blocks of linked loci within a genome

anonymous DNA polymorphisms

differences in genomic DNA sequence with no effect on gene function/phenotype

Protein Sequence identity

exactly the same amino acids in the same position

polymorphism

sequence differences between two individuals of the same species

single nucleotide polymorphism

variation in a DNA sequence occurring when a single nucleotide in a genome is altered

Southern blot

A *DNA* sample is electrophoresed on a gel and then transferred to a filter. The filter is then soaked in a denaturant and subsequently exposed to a labeled DNA probe that recognizes and anneals to its complementary strand. The resulting ds labeled piece of DNA is visualized when the filter is exposed to film.

Copy number variants (CNVs)

----Represent quantitative differences in number of large DNA sequences ---Mechanism that can produce new alleles = unequal crossing-over. During meiosis I, tandem arrays of the repeating units on homologous chromosomes can pair out of register. If recombination takes place btw mispaired repeating units, gametes are produced that have more or fewer copies of repeating unit than the originals

Positional cloning

--using SNPs and pedigree analysis to find the genomic location of typical alleles --object is to obtain info about unknown location of disease gene by finding polymorphic loci to which the mutation is genetically linked ---B/c we know from the human genome sequence the exact position of each locus, discovering anonymous DNA polymorphisms closely linked to the disease gene allows researchers to focus their search for the mutation on a small region of a single chromosome.

Alkaptonuria

A disease caused by an inability to degrade phenylalanine -a mutation in homogentisicaciddioxygenase (HGO) --Mendelian recessive and an early indication that mutations cause defects in enzymes

Taq polymerase

A heat-stable form of DNA polymerase extracted from bacteria that live in hot environments, such as hot springs, that is used during PCR

Huntington's disease

A human genetic disease caused by a dominant allele; caused by trinucleotide repeat CAG ---more repeats = earlier onset ---amplify CAG containing part, measure length using PCR product, can determine how many CAG repeats

whole genome sequencing

A method of genome sequencing that selects clones at random from a genomic library and after sequencing them, assembles the genome sequence by using software analysis --used to identify disease causing mutants

Polymerase Chain Reaction

A method of producing thousands of copies of DNA segment using polymerase 1)Heat to denature & separate strands, add primer & nucleotides 2) Add Polymerase: synthesize 5' to 3' 3) Rxn is eponential starting from 1st strand

no effect

As with all other polymorphisms, most SSRs occur outside the coding regions of genes and have ________on phenotype. HOWEVER SSR variations within genes can have profound phenotypic consequences. Ex: long tracts of trinucleotide repeats are the molecular cause of several severe neurological conditions, including fragile X syndrome and Huntington disease.

ChIP procedure

Association btw a protein of interest & genome can be mapped using (ChIP). 1) Following chemical protein-DNA crosslinking, the protein + associated DNA is purified 2) DNA sequence can be determined using next-Gen sequencing (i.e., ChIP-Seq) or micro array analysis (ChIP-chip)

double heterozygote

Basic requirement for genetic mapping is that at least one parent must be a _______. Figure 11.21b emphasizes this crucial point by showing that if neither parent is a double heterozygote, the cross cannot be informative.

mRNAs

Because cDNAs are made from ________________ they lack introns and they also do not have regulatory region information such as promoters and enhancers. --Clones in a cDNA can include 5′ UTR and 3′ UTR sequences. The reason is that the 5′ UTR and 3′ UTR sequences are found in exons, and the cDNA clones contain these exons

transcribed

Clones of a cDNA library represent only the fraction of the genome that is _________ in that tissue. The frequency with which particular fragments appear in a cDNA library is proportional to the level of the corresponding mRNA in that tissue. ---purpose of cDNA library is to find the transcribed regions (genes) ---clones of a genomic library represent every locus an equal number of times

chromosomal rearrangements

Cutting and reassembling of chromosomal blocks accompanying evolution --translocations connect part of one chromosome to part of a different, nonhomologous chromosome. ---- inversions flip a region of a chromosome 180° with respect to the rest of the chromosome. --- The farther back in time two species last shared a common ancestor, the more chromosomal rearrangements that alter the order of genes accumulate in each separate lineage

allelic heterogeneity

Different mutations in the same locus produce the same phenotype ---genetic diseases can be caused by a variety of different mutations in the same gene ex: cystic fibrosis

Next Generation Sequencing

Entire genomes sequenced using multiple parallel reactions to analyze short segments of DNA and compare the results to known sequences. --Key to the automated process -the 4 different dideoxy NTP have different color fluorescent tags --all 4 reactions are mixed together and separated by electrophoresis. The color and relative position of each fragment is monitored by an appropriate laser and detector as it runs off the gel, providing the order of nucleotide in the fragment

restriction enzymes

Enzyme that cuts DNA at a specific sequence of nucleotides by cleaving bonds in sugar phosphate backbone --recognizes a specific sequence of bases anywhere within the genome and then severs two phosphodiester bonds at that sequence, one in the sugar-phosphate backbone of each strand

form colonies

Example -transformation of a cloning experiment using pUC19 --Mixed together target DNA fragment & pUC19 plasmid DNA cleaved w/ same restriction endonuclease, then add ligase ---- end up w/ mixture: some pUC19 plasmids will now have combined w/ target fragment, many pUC19 molecules will not have combined ----To find plasmid cells: Plate the E. coli transformation on rich media containing ampicillin, X-gal, and IPTG. ----Because of the ampicillin,only cells *containing a plasmid* will ________________ ----Colonies that lack an insert with be blue, colonies containing an insert will be white.

lacZ, X-gal, white

Example of a common plasmid vector -pUC19 --origin of replication that works in E. coli (ori), &gene that makes host resistant to antibiotic Ampicillin (bla) ---Polylinker site is included: ------contains DNA sequence cleaved by common restriction endonucleases ----Polylinkersite is in frame w/ lacZ coding region. ----If no DNA fragment is cloned into the polylinker, ______ is made and the colony is blue on plates containing ____________, in contrast, inserting a DNA fragment in the polylinker will disrupt the lacZ reading frame and generate _________ colonies on X-gal media

alternative splicing

Exons and introns in the primary transcript can be ______________often in cell type- specific ways; as a result, the same gene can express different proteins. Researchers analyze alternative splicing by sequencing multiple cDNA clones from libraries made from each of many different tissues. --cDNA sequences will reveal which exons appear in processed mRNAs in particular cell types, and will predict amino acid sequences of proteins present in those tissues

Protein Sequence homology

General term indicating evolutionary relatedness among sequences sequences are homologous if they are derived from a common ancestor sequence Often discussed as percent homology --regions that are highly conserved are likely to be functionally important, region less well conserved tend to be less functionally important

Gene splicing

Generating a specific DNA sequence, usually assembled from various pieces, to either test a scientific idea (how a gene is regulated) or to make large amounts of either a specific protein or nucleic acid sequence.

Orthologous genes

Genes in two different species that arose from the same gene in the species' common ancestor; usually but not always retain the same function

polymerase chain reaction (PCR)

Goal is to amplify a target region of DNA. -----Two 16- to 30-base-long oligonucleotides, the PCR primers, define the ends of the target region. ------synthesize these primers based on prior knowledge of the genome. ----One oligonucleotide is complementary to one strand of DNA at one end of the region; the other oligonucleotide is complementary to the other strand at the other end of the region. If these primers are drawn as 5′→3′ arrows to indicate their polarity, the arrows will point toward each other through the target region

gel electrophoresis

How do you confirm that you have built the desired genetic construction? Fragments generated by different restriction endonucleases are diff. sizes, use gel electrophoresis to ensure have generated correct fragments b/c DNA molecules of different size migrate with different speeds in an electric field

hybridization

Identical or very similar DNA sequences can be identified through base pairing of a labeled nucleic acid probe to denatured DNA in a process called _________________

confirm

In order to ___________if you have desired clone, use restriction endonuclease digest and gel electrophoresis ex: inserted 1200 bp, find 1200 band in gel

Gene desert

In reference to the density of genes in the genome, a region that is gene poor—that is, a long stretch of DNA possibly consisting of hundreds of thousands to millions of base pairs completely devoid of any known genes or other functional sequences.

compound heterozygote

Individual with two different mutations in same gene --two different recessive loss-of-function mutations in the same gene may display the mutant phenotype. An individual with two different recessive alleles at a locus that results in a recessive phenotype.

Simple Sequence Repeat

Individuals have extensive DNA variation with one difference in every 1000bp ---_________ are highly polymorphic and are often used for DNA fingerprinting --number of repeat units varying greatly among different members of the population (i.e., many different alleles). The high level of variation can be used to specifically identify someone by the specific SSR alleles they contain --Most of the SNPs are between genes or in introns and do not cause phenotypes

two point cross

Instead of dealing with only two or three loci at a time (as in a two-point or three-point cross), you can use DNA microarrays to follow millions of anonymous loci in each person in the pedigree. You can think of this microarray/pedigree analysis most simply as the simultaneous performance of millions of __________, each one a test for linkage between an individual DNA marker and the disease locus. Discovery of a DNA marker that shows linkage to the disease locus is the first goal of positional cloning.

whole-exome sequencing

Investigators first enrich (by hybridization to cDNA sequences) for genomic DNA fragments that correspond to the exons of all genes, and then sequence these fragments --exome = collection of all exons of all genes

Immunoprecipitation (IP)

Involves precipitating an antigen out of solution using an antibody as a purification step 1) antibody added & bind protein of interest 2) Protein added to make antibody protein complex insoluble 3) Centrifugation of solution pellets antibody-protein complex, remove supernatant

exons

It is important to note that this cDNA library includes only _________from part of the genome that these cells were actively transcribing for translation into protein. The clones in cDNA libraries do not contain introns because the mature mRNAs from which they were produced do not have introns.

fragments, amplify

Key to Genetic cloning/splice expts include 1.the ability to make very specific DNA ______________ (using restriction endonucleases, polymerase chain reaction (PCR), or direct DNA synthesis) and join them precisely together (e.g. DNA ligase) 2.The ability to _________________ your construct (usually by growing it in E. coli) and verify that what you built is correct (e.g., restriction digests + gel electrophoresis or DNA sequencing) 3.Eventually using your genetic construct to test a question or make lots of protein (e.g. Western blotting)

Western blot

Laboratory method used to detect specific protein molecules from among a mixture of proteins. This mixture can include all of the proteins associated with a particular tissue or cell type. Western blots can also be used to evaluate the size of a protein of interest, and to measure the amount of protein expression.

stop codons

Look for potential genes by finding open reading frames --Reading frames that truly encode proteins will be "open" meaning that there are no stop codons until the end; reading frames that do not encode proteins will have many __________________ ---Sequence conservation among organism for a predicted gene helps suggest that it is actually important ad expressed.

exon shuffling

Mechanism for the evolution of new genes; in the process, coding sequences from different genes are brought together to generate a protein with a novel combination of domains.

mixture of different sequences

Mirroarray is basically the same idea as a Southern blot -one hybridizes a fluorescent DNA probe against array, and determine which spots on the microarray become fluorescent (indicating the probe bound to the specific sequence on the microarray) ---Difference: With a Southern blot probe, the probe only represents 1 unique piece of DNA(e.g. part of a gene). ----With a microarray experiment, the probe is a (often very complex) _____________________

candidate genes

Positional cloning identifies DNA polymorphisms that are linked to disease genes. After a disease gene is mapped approximately, researchers sequence_____ in the region to identify one that is altered consistently in affected individuals.

gel electrophoresis

Procedure used to separate and analyze DNA fragments by placing a mixture of DNA fragments at one end of a porous gel and applying an electrical voltage to the gel ---DNA has negative charge, will move toward positive charge ---agarose gel forms a maze of obstructions so larger DNA fragments slowed down

exon/intron structure

Purpose of having both cDNA library & genomic library: Determine sequences of many cDNA clones, and compare these cDNA sequences with that of the genome. Regions of identity between cDNA and genome represent the exons of genes, and the sequence of a complete cDNA (copied from a full-length mRNA) allows you to determine the______ of the corresponding gene.

cDNA clones

Reverse transcriptase produces complementary DNA (cDNA) from mRNA transcripts; _________thus represent only the exons of genes.

fluorescent spot

SNP Microarray procedure 1) Make a fluorescent probe that contains the entire genome 2) Hybridize this genomic probe against a microarray that contains oligos that represent all of the known SNP in the organism ----The ___________-each correspond to SNP in the probe sample

mismatch

SNPs can also be detected by nucleic acid hybridization because a single____________ between a short DNA probe and a sequence makes the hybridization less stable --A Mismatches between the probe and the target lowers the melting temperature (Tm) of the resulting DNA duplex Tm = temperature are which 50% of the Duplex DNA molecules dissociate

slipped mispairing

SSRs arise spontaneously from rare, random events that initially produce a short repeated sequence with four to five repeat units. Once a short SSR mutates into existence, however, it can expand into a longer sequence by a form of faulty DNA replication called _________ or stuttering

SSR

SSRs can be detected by PCR followed by sequencing or by PCR followed by size analysis on a gel ---SNPs detected by PCR then sequencing

mispairing

Some diseases are caused by triplet repeat SSRs Example -Huntington disease SSRs expand and contract due to slipped ____________ during DNA replication

gene families

Some genes are part of ________________ where a group of genes are closely related in sequence and function ---evolve when a gene is duplicate and then diverges from the ancestral gene --Examples of gene families include the genes that encode the hemoglobins that allow us to transport oxygen in our blood, the immunoglo- bins (antibodies) that help us ward off infections, and the olfactory receptors critical for our sense of smell.

Protein Sequence Similarity

Substituting one amino acid for another with very similar properties. (e.g., serine and threonine are usually considered very similar amino acids)

transcribed

The Most Direct Method to Find Genes Is to Locate __________Regions --all genes are transcribed into RNAs, even if some RNAs are not translated. --most mRNAs are so relatively rare in cells that they cannot be purified readily --easiest way to study mRNAs is to copy them into DNA, to clone the resultant DNA molecules, and then to sequence these clones by the same methods already described for genomic DNA

reversible protein-DNa crosslinking

The entire ChIP procedure depends upon _____________________ a system in which the protein/DNA interaction can be stabilized during purification, then neutralized later to facilitate purification of the DNA from the protein. -----Accomplished by treating the cells with formaldehyde, which generates covalent protein-DNA crosslinks that can be easily reversible upon removal of free formaldehyde and incubation and elevated temperatures.

Colony hybridization

The identification of a colony containing a desired gene by using a DNA probe that is complementary to that gene. --transfers the colonies from the library transformation (i.e., blots he colonies in a Petri dish) to a membrane rather then DNA separated by electrophoresis ---variation of Southern blot

spliced

The main reason geneticists studying eukaryotic cells make cDNA libraries is to determine how a gene's primary transcript is ____________ in a given cell type. Almost all eukaryotic genes are interrupted by introns. In contrast, the genomes of bacteria do not have introns and the large majority of the base pairs in bacterial genomes are protein-coding regions

oligo dT

The primers used in creating cDNA are also_______so as to initiate polymerization of the first cDNA strand from the 3′ ends of all mRNAs. 1) After synthesis is finished, you can denature mRNA-cDNA hybrids into single strands by heating the hybrids to high temperature. 2) Add RNase enzyme that digests original RNA strands leaves intact single strands of cDNA which fold back on themselves at their 3′ ends to form hairpin loops that serve as primers for synthesis of the second DNA strand w/ DNA polymerase 3) The products are double-stranded cDNA molecules.

domain architectures

The shuffling, addition, or deletion of exons during evolution can create new genes whose protein products have novel _______ (different numbers and kinds of do- mains in different orders) and thus can assume new roles in cells and organisms.

sequencing

The technique of _________ PCR products amplified from genomic DNA is a straightforward way to determine one's genotype for any SNP. ---possible to genotype a polymorphism in a PCR product without actually sequencing it. Gel electro- phoresis can easily distinguish small variations in the actual size of a locus caused by DIPs or SSRs,

annotate

To ________________ : identify locations of genes and all of the coding regions in a genome and determining what those genes doa genome, you would have to sequence many clones from many cDNA libraries because different genes are transcribed in different tissues and because some genes are transcribed only rarely in any tissue.

Co-Immunoprecipitation (co-IP)

Used to determine binding partners of your target protein ------an antibody is used to purify its target antigen, along with its binding partners, from a mixed sample. In this case, the antigen is the bait protein and its binding partners are the prey proteins that are co-purified by the antibody:antigen interaction.

Microarray

Used to test SNPs across the genome in single expt --small glass plate w/ thousands of spots -each spot contains the DNA from a different GENE, and location/ identity of each spot is known. ---represents essentially all of the information from a genome on a single slide

polymorphic

When two or more alleles exist at a DNA locus, the locus is __________, and the variations themselves are DNA polymorphisms (DNA markers). Most polymorphisms are anonymous; they have no effect on phenotype.

restriction enzyme, vector

You know how to design the primer for DNa sequencing because: (1) You added the insert into the vector at a known location defined by a specific ______________________ and (2) you know the sequence of the________________that flanks the position of the insert.

DNA Fingerprinting

___________Examines Multiple SSR Loci --polymorphism of SSR loci makes them a powerful resource in identifying a person from his or her DNA. The power comes from the possibility of examining multiple polymorphic SSR loci simultaneously: possibility of 2 people having SSR share exactly the same combination of two alleles of a particular SSR is extremely low unless identical twins

duplication and divergence

each gene family evolved by a process of _________from an ancestral gene. The two DNA sequence products of a duplication event, which start out identical, eventually diverge as they accumulate different mutations (

pseudogenes

former genes that have accumulated mutations and are nonfunctional

de novo genes

genes lacking homologs except in closely related species ---young genes that evolved recently from ancestral intergenic sequences --evolution through mutation: Either transcribed intergenic regions gained an ATG and thus a short ORF or small intergenic ORFs that were not originally transcribed acquired transcriptional regulatory sequences

poly A tail

mRNAs constitute only a small fraction of all the RNAs in the cell so want to separate mRNAs from abundant rRNAs and tRNAs. -----Possible b/cmRNAs in eukaryotic cells have _________at their 3′ ends so mRNAs will hybridize through their poly-A tails to the oligo-dT magnetic beads ---addition of reverse transcriptase to this total mRNA—& 4 deoxyribo nucleotide triphosphates and primers to initiate synthesis— generates single-stranded cDNA bound to the mRNA template

locus heterogeneity

mutations in one of two or more different genes can cause the same disease.

exome

part of the genome corresponding to the exons of all known genes