Exam 3 - Genetics

if ddNTPs are included in a DNA synthesis reaction, the polymerase will occasionally _______.

(randomly) insert a ddNTP instead of a dNTP into a growing DNA strand. This occurrence causes synthesis to terminate because DNA polymerase cannot add new nucleotides to a ddNTP due to it lacking a 3' oxygen

Cloning DNA with a plasmid vector

- Plasmid DNA and DNA to be cloned are cut with the same restriction enzyme - DNA restriction fragments from DNA to be cloned are added to a linearized vector in the presence of DNA ligase. - Sticky ends anneal and DNA ligase creates bonds to seal nicks in DNA backbone -Produces recombinant DNA and introduces it into bacterial host cells

Probes

used to screen library and recover clones of specific gene

Library Screening

used to sort through a library and isolate specific genes of interest

Applications of PCR

useful and widely used tool -DNA cloning using PCR-based technique had advantages over library cloning -PCR is rapid taking a few hours -very sensitive and amplifies specific DNA sequences from small DNA samples -used in genetic testing, forensics, and molecular pathology -also key method for screening mutations involved in genetic disorders. also detecting bacteria and viruses

Producing cDNA:

uses technique of reverse transcription 1.enzyme reverse transcriptase uses mRNA as a template to synthesize a complementary DNA (cDNA) and forms a couple-stranded mRNA/cDNA duplex 2.mRNA is then partially digested with the enzyme RNAase H to produce gaps in the RNA strand 3.3' ends of remaining mRNA serve as primers for DNA polymerase I, which synthesizes a second DNA strand. 4.results are double-stranded cDNA molecule that can be cloned into suitable vectors, usually plasmids

changes in DNA sequences give rise to _________.

variations that result in phenotypic variability, adaptation to environmental changes, and evolution

Solutions to CRISPR-Cas limits

-modify Cas9 to improve specificity -improve sgRNA design via algorithms -find alternative enzymes from bacteria or archaea

Mechanisms for repairing double-stranded (ds) DNA breaks:

1. nonhomologous end-joining (NHEJ) 2. homology-directed repair (HDR)

PCR requires:

1.double-stranded target DNA 2.DNA polymerase 3.Mg2+ (DNA polymerase cofactor) 4.Four deoxyribonucleoside triphosphates 5.Primers (short, single-stranded sequences; one complementary to 5' end and another the 3' end)

Fluorescence in situ hybridization (FISH)

A method of in situ hybridization that utilizes probes labeled with a fluorescent tag, causing the site of hybridization to fluoresce when viewed using ultraviolet light. -hybridizing probe directly to chromosome or RNA without blotting -carried out with isolating chromosomes on slide or in situ tissue section or entire organisms -helpful when embryos are used for various studies in developmental genetics -can be used to produce special karyotypes

electroporation

A technique to introduce recombinant DNA into cells by applying a brief electrical pulse to a solution containing the cells. The pulse creates temporary holes in the cells' plasma membrane, through which DNA can enter.

Bacterial artificial chromosome (BAC)

A type of large cloning vector; an artificial version of a bacterial chromosome that can accept DNA inserts 100-300 kb range -generally very large -low copy number (one or two per bacterial cell) plasmids

Yeast artificial chromosome (YAC)

A type of large cloning vector; that have telomeres at each end, origins of replication (ori), and a centromere -joined to selectable marker genes and to a cluster of restriction-enzyme recognition sequences for foreign DNA insertion -up to 1000 kb of DNA insert is possible

Why are restriction enzymes useful?

Able to accurately and reproducibly cut DNA into fragments

Nonhomologous end joining (NHEJ)

An error-prone mechanism of double-stranded DNA break repair in eukaryotic genomes in which damaged nucleotides are removed and blunt ends of strands are joined.

transgenic animals (knock-in animals)

Express or overexpress particular gene of interest (transgene) Vector with transgene undergoes homologous recombination into host genome Vector with transgene can also be put into ES cells and injected into embryos Allows for study of effects on appearance and function in mice

How are mutations classified?

By molecular change

Bacterial cells with recombinant (w/ plasmids containing DNA fragments in lacZ gene) plasmid

FORMS WHITE COLONIES; functional beta-galactosidase cannot be made

basis of gene function:

DNA molecule stores, replicates, transmits, and decodes information

Recombinant DNA

DNA produced by combining DNA from different sources

Bacteria production of restriction enzymes

Defense mechanism against infection by bacteriophage restricting/preventing viral infection by degrading the DNA of the invading viruses

Palindrome

Form of symmetry exhibited by recognition sequence where the nucleotide sequence reads the same on both strands of the DNA when read 5' to 3'

Most common recognition sequence:

Four or six nucleotides long

Why is process of blue-white screening mechanism done?

Not all plasmids will incorporate DNA to be cloned and the recombinant ones need to be identified

Quantitative real-time PCR (qPCR)

Real-time PCR; uses fluorescent probes to quantitate the amount of DNA or DNA product after each round of amplification -able to quantify PCR product as it is made during the experiment without having to run a gel -allows calculation of amount of template DNA originally present in a sample

Reverse transcription PCR (RT-PCR)

Methodology for studying gene expression (mRNA production by cells or tissues) Reverse transcriptase is used to generate ds-cDNA -following generation of cDNA, PCR is sued to amplify cDNA with a set of primers specific for the gene of interest -amplified cDNA fragments are then separated and visualized on an agarose gel -amount of amplified cDNA in RT-PCR is based on relative number of mRNA molecules in the starting reaction -useful for evaluating relative levels of gene expression

Blunt ends

Restriction fragments with no overhanging ends (such as AluI) -can be joined to ea other by DNA ligase, but they are harder to ligate because there are no single-stranded overhangs holding DNA molecule

Western Blotting

Used for analyzing proteins -proteins separated by gel electrophoresis

donor template

Used for genome editing by CRISPR-Cas; an experimentally introduced DNA molecule carrying a sequence with desired edits flanked by "homology arms" with sequences that match regions of the genome adjacent to the target sequence to be edited.

Cloning Vectors

a DNA molecule that can accept foreign DNA, be transferred to a host cell, and get replicated in it -have several restriction enzyme sites to allow insertion of DNA fragment -carry selectable gene marker/reporter gene

Expression vectors

a DNA vector that is designed to ensure mRNA expression of a cloned gene with the purpose of producing many copies of the encoded protein in host cell -available for both bacterial and eukaryotic host cells -Ti plasmid and soil bacterium can be used for introducing genes in plants

point mutation/base substitution

a change of one base pair to another in DNA molecule

missense mutation

a change of one nucleotide of a triplet within a protein-coding portion of a gene resulting in the creation of a new triplet that codes for a different amino acid in protein product

DNA sequencing site

allows for identification of DNA segment into cloned plasma

Non Transformed bacteria do not have (blue-white screening_

ampR

Each cycle of PCR results in __________.

amplification, doubling the number of DNA molecules present at the start of the cycle

recognition sequence/restriction site

Specific DNA nucleotide sequence where a restriction enzyme binds; cuts both strands of DNA within that sequence by cleaving the phosphodiester backbone; "digestion" of DNA

Bacterial cells with nonrecombinant (self-ligated; no inserted DNA) plasmid:

TURNS BLUE; functional enzyme cleaves x-gal medium

mutations

an alteration in the nucleotide sequence in a genome, any base-pair change in sequence, single base-pair substitution, deletion or insertion of base pairs, major alteration in chromosomal structure -can occur in coding/noncoding regions, regulatory sequences -promoters, enhancers, splicing signals

What is a probe?

any DNA or RNA sequence complementary to target gene of sequence being identified -probe must be labeled or tagged (chemical or color reactions)

single molecule sequencing in real-time (SMRT)

attaches single molecule of DNA polymerase anchored to substrate -visualizes in real time the syntheses of a strand of DNA by polymerase

third-generation sequencers (TGS)

based on sequencing a single molecule of single-stranded DNA -SMRT: single molecule sequencing in real-time

Modification of plasmids

can be modified to serve as cloning vectors -one feature it contains is a region, multiple cloning site

computer-automated sanger-reaction

can produce large amounts of sequence DNA in a short period of time -enabled rapid progress of human genome project

DNA Libraries

collection of cloned DNA in host cells; can be screened to identify particular gene of interests -depending on construction may contain genes and noncoding region of DNA -two main types: genomic DNA libraries and complementary DNA (cDNA) libraries

Complementary DNA (cDNA) Libraries

collection of cloned cDNA sequences -offers certain advantages over genomic libraries -cDNA libraries contain DNA copies, cDNA -represents only genes that were expressed at the time library was made (useful for identifying genes involved in cancer formation)

Genomic Libraries

collection of clones that contains all the DNA sequences of an organism's genome -consists of many overlapping fragments of the genome -has at least one copy of every DNA sequence in organism's chromosomes -constructed by extracting chromosomal DNA and cutting genomic DNA with restriction enzymes and ligating fragments into vectors -libraries built from eukaryotic cells will contain coding and noncoding segments such as introns

Northern blotting

commonly used to study gene expression (RNA production) by cells and tissues -it could both characterize and quantify the transcriptional activity of genes -A technique that enables specific nucleotide sequences to be detected in samples of mRNA. It involves gel electrophoresis of RNA molecules and their transfer to a membrane (blotting), followed by nucleic acid hybridization with a labeled probe.

cDNA

complementary DNA that are made from mRNA molecules isolated from cultured cells or tissues -synthesizing complementary DNA using reverse transcription

The key to the Sanger technique is the addition of ______ to terminate the chains of nucleotides.

dideoxynucleotides (ddNTPs)

blue-white screening mechanism

identify transformed cells containing recombinant or nonrecombinant plasmids; plasmid is used that contains lacZ gene into which a multiple cloning site has been incorporated -if inserting a DNA fragment anywhere in multiple cloning site disrupts lacZ gene, it won't produce function beta-galactosidase -agar plates used containing antibiotic ampicillin; also contains X-gal (lactose analog) -x-gal turns blue when cleaved by the enzyme

What do selectable gene markers/reporter genes do?

make it possible to distinguish between host cells that have taken up vectors and host cells that have not

Gene targeting

method for altering the sequence of a specific gene by introducing the modified version on a vector -target specific allele, locus, or base sequence and learn its function on gene of interest

Agarose Gel Electrophoresis

method of DNA analysis that separates DNA fragments by electrical currents; separates them by size -smallest pieces move farthest through gel -fragments form a series of bands, visualized by treating gel with DNA-binding stains (like ethidium bromide) and illuminating with UV -mainly used when smaller pieces of DNA need to analyzed or isolated

CRISPR-Cas9

most widely used crispr-cas system for genome editing uses a nucleus, Cas9 from bacterium Streptococcus pyogenes -cas9 has two nuclease domains and can create a double-strand break in DNA -only cuts DNA near specific sequence, protospacer adjacent motif (PAM) 5'NGG-3' -Cas9 requires single guide DNA (sgRNA) for activity and specificity

plasmid

naturally occurring, extrachromosomal, double-stranded, circular piece of genetic material found in bacteria that can replicate separately from chromosomes within bacterial cells -first vectors developed

chimeras

organism made up of cells from two or more individuals

genome editing

removing, adding, or changing specific DNA sequences in genome of living cells; techniques create double-stranded breaks in the DNA double helix, enabling insertion of a desired DNA sequence or removal of a sequence

Next generation sequencing (NGS)

second-generation after sanger methods; simultaneous reaction synthesize DNA from tens of thousands of identical strands -used fluorescent imaging techniques to detect new strands Faster, more recent sequencing methods that are continually developing and becoming more efficient, powerful and cost effective.

Using Crispr-Cas9 to disrupt gene function or correct mutation:

takes advantage of eukaryotic cell's double-strand DNA break mechanisms -Cas9 and sgRNA *single guide* are introduced into cells in order to disrupt gene function or create nonfunctional allele

Limitations of PCR

-information about nucleotide sequence of target DNA is required to synthesize primer -sensitivity: minor contamination from other sources can cause (e.g., skin cells from researcher) -cannot typically be used to amplify long segments of DNA

Limitations of CRISPR-Cas

-can cut at off-target sites in genome -sgRNA may have more than one perfect match -sgRNA may direct Cas9 to sequences with one of few mismatches

Diverse applications of CRISPR-Cas9

-creation of tomatoes that ripen quickly -"bringing back" woolly mammoth -fight off livestock diseases -modify food crops for additional nutritional traits, pest, and drought resistance -gene therapy -clinical trials for cancer

Embryonic (ES) stem cells

-using ES cells, scientists introduce targeting vectors into cells via electroporation -ES cell takes in targeting vector, and endogenous enzyme recombinase catalyzes homologous recombination -recombinant ES cells are injected into mouse embryo -results in chimeras

Two main techniques of transformation

-using calcium ions and brief heat shock to pulse DNA into cells -electroporation

Knockout Mice (KO)

1. designing of targeting vectors, creates segment of DNA for introduction into cell 2. targeting vector then undergoes homologous recombination with gene of interest and renders it nonfunctional 3. target vector has mutated a copy of gene of interest 4. foreign DNA disrupts the reading frame and produces nonfunctional protein

Clones

recovered copy of a recombinant DNA molecule

What two tools of recombinant DNA technology are used to construct and amplify DNA molecules?

1. Restriction enzymes (DNA cutting enzymes) 2. Cloning Vectors

X-gal

A Chemical similar to lactose that turns dark blue when cleaved by beta-galactosidase

reporter gene

A genetic marker encodes a protein that produces a visible effect, such as color or fluorescent light -assessment of enzymatic activity -protein reporting

Southern blotting

A hybridization technique that enables researchers to determine the presence of certain nucleotide sequences in a sample of DNA; identify number of copies of particular sequence or gene present in genome -DNA fragments produced by restriction enzyme digestion are separated by electrophoresis and transferred (blotted) to a DNA binding membrane, and hybridization of these fragments to a labeled DNA or RNA probe -Membrane washed to remove excess probe -overlay of x-ray film for autoradiography -only fragments hybridized to probe are visible

CRISPR-Cas

Clustered Regularly Interspaced Short Palindromic Repeats -initially discovered to understand how bacteria fight viral infection and later as genome-editing tool

Restriction enzymes

Enzyme that cuts DNA at a specific sequence of nucleotides.

Homology directed repair can be tricked

It can be "tricked" into using artificial donor template (instead of the homologous chromosome) to make complex substitutions, deletions, or additions

Cohesive "sticky" ends

Overhanging single-stranded ends of a DNA molecule created by the action of certain restriction enzymes (such as HindIII)

By copying a specific DNA sequence through a series of in vitro reaction:

PCR can amplify target DNA sequences that are initially present in very small quantities in a population of other DNA molecules

Technique developed to accelerate method of DNA cloning:

Polymerase chain reaction (PCR)

spectral karyotype

Probes fluoresce different colors onto chromosomes to make an easier detection for defective chromosomes

anneal

The joining together of DNA fragments by hydrogen bonding

Three steps of PCR

Three reaction steps in a cycle: 1. Denaturation: double-stranded (ds) DNA to be cloned is denatured into single strands by heating it 2. Hybridization/Annealing: Temperature of reaction is lowered to allow primer binding (hybridization/annealing), to the denatured, single-stranded DNA; starting point for DNA polymerase to synthesize new DNA strands complementary to the target DNA 3. Extension: Reaction temperature is adjusted, and DNA polymerase uses the primer as a starting point to synthesize new DNA strands by adding nucleotides to the ends of the primers (5' --> 3')

Gene knockout (KO)

a genetic technique in which one of the genes of an organism is "switched off"; the term can also be used to describe the organism that carries this inoperative gene -loss-of-function mutation -KO mice revolutionized mice

Blue-white screening

a method for determining whether bacterial cells have incorporated a plasmid that includes a gene of interest; identifies cells containing recombinant and nonrecombinant DNA

Loss-of-function mutation

a mutation that produces alleles encoding proteins with reduced or no function

Dideoxynucleotides (ddNTPs)

a nucleotide used in DNA sequencing, it has a 3' hydrogen (H) instead of a 3' hydroxyl (OH) If a dideoxyribonucleotide is incorporated into a DNA strand, it stops any further growth of the strand. -called chain-termination nucleotides because they lack the 3' oxygen required to form a phosphodiester bond with another nucleotide

Polymerase chain reaction (PCR)

a rapid method of DNA cloning; method for amplifying DNA segments that depends on repeated cycles of denaturation, primer annealing, and DNA polymerase--directed DNA synthesis -extends power of recombinant DNA -eliminates need to use host cells for cloning -copies specific DNA sequence via in vitro reactions

EcoRI

a restriction enzyme that specifically cuts DNA with sequence GAATTC and creates sticky ends -dna fragments from EcoRI can base-pair w/ complementary single-stranded ends of DNA from different sources and can anneal together via hydrogen bonding-- DNA ligase added to DNA fragments seal the phosphodiester backbone of DNA to covalently join the fragments together to form recombinant DNA molecules

multiple cloning site

a short sequence with many restriction enzyme sites to facilitate the insertion of additional DNA fragments -region where pieces of DNA are cloned into plasmid

mutations provide basis for genetic analysis

act as "markers" for genes

Homology directed repair (HDR)

after double-strand break, cellular mechanism to use sister chromatids as template to replace are of break via homologous recombination only occurs during *mitosis* precise, but inefficient

Ori, origin of replication

enables replication of plasmid within bacterial cell; enhances number of DNA clones that can be produced

lacZ gene

encodes for beta-galactosidase; in DNA cloning plasmid, lacz gene, reporter gene allows for selection of bacterial cells that contain a cloned DNA fragment -another strategy for identifying recombinant DNA in cloning experiment

Dideoxynucleotide chain-termination sequencing (Sanger)

first and most common method of DNA sequencing (Fred Sanger) -a double-stranded DNA molecules whose sequence is to be determined is converted to single strands, one of which is used as a template for synthesizing a series of complementary strands -template DNA is mixed with a primer that is complementary to either target DNA or the vector, DNA polymerase, and the four deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP)

Ampicillin resistance gene (ampR)

for ampicillin resistance; in DNA cloning plasmid, ampR, selectable marker gene allows for selection of bacterial cells that contain the cloning plasmid

selectable marker gene

genes carried by plasmids for certain traits, often for antibiotic resistance

Phage vectors

genetically modified bacteriophage λ (double-stranded DNA viruses) -have multiple cloning site -Carry up to 45kb of cloned DNA

Important distinction between expression vectors and plasmids, phage vectors, and YACs:

plasmids, phage vectors, and YACs only CARRY DNA. They do not signal the cell to transcribe mRNA like expression vectors

Transformation

process by which plasmids are introduced into bacteria

Why is PCR considered a chain reaction?

products of previous cycle serve as templates for each subsequent cycle

nonsense mutation

triplet being changed into a stop codon, resulting in termination of protein translation