Unit 7 DNA & BIOTECH

The Polymerase Chain Reaction (cycle 1)

1. DENATURATION: heat briefly to separate DNA strands 2. ANNEALING: cool to allow primers to form hydrogen bonds with ends of target sequences 3. EXTENSION: DNA polymerase adds nucleotides to the 3' end of each primer

Griffith experiement:

4 experiments where he injected smooth or rouch strain into mice

Complementary DNA

A form of eukaryotic gene that only has exons because it is synthesized from mRNA.

Recombinant DNA

A molecule containing DNA from 2 different sources.

how might induced pluripotent stem cells resolve the debate about using stem cells for medical treatments

Because it differentiates regular human cells into embryonic stem cells, there is not taking of cells from embryos

The similarity between RNA interference and In vitro Mutagenesis.

Both disable genes in order to observe its function in a cell.

Which of the following processes would allow the detection of alternative splicing of transcripts from a given gene?

Compare the sequences of different mRNAs made from the given gene.

Solution to the presence of introns in Eukaryotic genes.

Complementary DNA - Reverse transcriptase synthesizes a DNA molecule using an mRNA template and the complementary DNA can be inserted into the cloning vector.

The difference between embryonic stem cells and adult stem cells.

ES cells are pluripotent and are harvested from an embryo while Adult stem cells can't give rise to all cell types like ES cells.

Restriction enzyme (restriction endonucleases)

Enzymes that protect the bacterial cell by cutting up foreign DNA from organisms or phages.

Solution to differences between bacteria and eukaryotes in promoters/control sequences

Expression vector: A cloning vector that contains a highly active bacterial promoter upstream of a restriction site. - eukaryotic gene can be inserted at the restriction site and the bacteria recognizes its promoter and expresses the eukaryotic gene.

True/False: Cloned genes can be expressed as protein in only bacterial cells.

False: Cloned genes can be expressed as protein in both eukaryotic and bacterial cells.

True/False: SNPs are directly involved with the disease.

False: SNPs show the most likely location for the gene that causes the disease.

Reason why Genome-wide association studies are used.

Genome-wide association studies are used because it is unethical to disable genes in humans for research purposes.

Large scale analyses of the genomes of a large group of people with a phenotypic condition or disease.

Genome-wide association studies.

What is the source of a viral envelope?

Host cell membrane -The viral envelope is derived from host cell membrane

Which of the following processes destroys RNA molecules in a cell if they have a sequence complementary to an introduced double-stranded RNA?

RNA interference

DNA Technology

Techniques for sequencing and manipulating DNA

How are bacterial artificial libraries and cDNA libraries formed?

To create a library using complementary DNA, researchers modify the cDNA by adding restriction enzyme recognition sequences at each end. Then the cDNA is inserted into vector DNA in a manner similar to the insertion of genomic DNA fragments. The extracted mRNA is a mixture of all the mRNA molecules in the original cells, transcribed from many different genes. Therefore, the cDNAs that are cloned make up a cDNA library containing a collection of genes. However, a cDNA library represents only part of the genome - only the subset of genes that were transcribed in the cells from which the mRNA was isolated.

Single Nucleotide Polymorphism

Variations at a single nucleotide.

Polymorphism

Variations in DNA sequence among a population.

Nucleic acid probe

ability to detect the mRNA by nucleic acid hybridization with molecules of complementary sequence that we can follow in some way -complementary molecule is short, single stranded nucleic acid called nucleic acid probe

Griffith 1928 tried to:

find a vaccine for Strep..

DNA methylation is a mechanism used by eukaryotes to do what? See Concept 18.2 (Page)

inactivate genes DNA methylation, involving the attachment of methyl groups to certain bases, is a mechanism for the long-term inactivation of genes during development.

When taken up by a cell, which of the following molecules binds to a repressor so that the repressor no longer binds to the operator?

inducer

Hershey/ Chase Experiment:

inserted prtein tagging t2 and e coli were grown in radioactive sulfer. Dna tagging t2 and e coli were drown in radioactive phosphorus. RESULT: the DNA labeled infected with e coli while the protein labeled was found in supernatant with the virusus

Which of the following types of DNA make up the rest of the human genome?

non-protein-coding DNA that is transcribed into several kinds of small RNAs with biological function

In E. coli, to repair a thymine dimer by nucleotide excision repair, in which order do the necessary enzymes act?

nuclease, DNA polymerase, DNA ligase

This is an image of a(n) _____. nucleotide thiol nucleic acid

nucleotide

Taq(DNA) polymerase

unstable bacteria, faster in head because kinetic energy

Chargaff did:

used chromatography to seperate nitrogenous bases

In Situ hybridization process

- A complementary DNA sequence is synthesized from the template strand creating the nucleic acid probe. - A fluorescent molecule is added to the nucleic acid probe. - the probe can hybridize with any complementary sequences on mRNAs in embryonic cells.

How restriction enzymes and PCR work together in gene cloning.

- PCR primers are designed with restriction sites matching ones in the cloning vector. - PCR produces DNA fragments that can be cut by the restriction enzymes, incorporated into plasmids and cloned. - The cloned gene is sequenced and the inserts without errors are chosen.

making cDNA from eukaryotic genes (five steps)

1. reverse transcriptase is added to a test tube containing mRNA isolated from a certain type of cell 2. reverse transcriptase makes the first DNA strand using the mRNA as a template and a stretch of dT's as a DNA primer 3. mRNA is degraded by another enzyme 4. DNA polymerase synthesizes the second strand, using a primer in the reaction mixture 5. the result is cDNA, which carries the complete coding sequence of the gene but no introns

Cytosine makes up 42% of the nucleotides in a sample of DNA from an organism. Approximately what percentage of the nucleotides in this sample will be thymine?

8 percent

Totipotent

A cell that has the potential to produce an complete new organism (ex: carrots) embrotic

cAMP receptor protein (CRP)

A regulatory protein that directly stimulates gene expression.

Restriction Site

A short DNA sequence 4-8 nucleotides long that the restriction enzyme recognizes and cuts at precise points.

Organismal cloning

Cloning that produces an organism genetically identical to the parent that donates the single cell.

Transformation

DNA from one strain of bacteria is assimilated by another strain. DNA from pneumonia-causing bacteria is mixed with harmless bacteria. The bacteria are injected into mice. The mice develop pneumonia and die.

Hershey and Chase 1952 discovered:

DNA is the genetic material for bacteriophages (used radioactive isotopes) -DNA has phosphorous -Protein has sulfer

In E. coli, which enzyme catalyzes the elongation of a new DNA strand in the 5' → 3' direction?

DNA polymerase III

PCR Process

Denaturation: Heated to separate DNA strands. Annealing: Cooled to allow primers to form hydrogen bonds with ends of the target sequence. Extension: Taq polymerase adds nucleotides to the 3' end of the primer. - fast - specific because of the primers specific to the sequences being amplified. - only a small amount of DNA is necessary. - Crime scene evidence. - Rapid prenatal diagnosis of genetic disorders - Detection of viruses usually difficult to detect.

Materials needed for PCR

Double-stranded DNA, all four nucleotides, 15-20 nucleotide DNA strands (primers) specific to the sequence being amplified, Taq polymerase

True/False: The older the donor nucleus the greater chance of a multicellular organism developing from the recipient cell.

False: The older the donor nucleus, the smaller the chance of a multicellular organism developing from the recipient cell.

Induced pluripotent stem (iPS) cells

Fully differentiated cells that are transformed back into ES cells becoming pluripotent again. embrotic and adult cells

DNA microarray assays

Gene tracking technology used to look at all mRNAs in a given tissues or in comparision of tissues -picture: glass slide with a large number of single stranded cDNA fragments

Technologies used for tracking/analyzing gene expression

In Situ Hybridization Reverse Transcriptase-Polymerase Chain Reaction DNA Microarray Assays RNA sequencing

Gene tracking technology used in order to detect the position of mRNA in an intact organism.

In situ hybridization

The difference between RNA interference and In vitro mutagenesis.

RNA interference uses synthetic double-stranded RNA molecules that match the sequence of a gene to block the translation or trigger breakdown of the gene's mRNA. In vitro mutagenesis introduces mutations into a cloned gene that can disable it and inserts the cloned gene into the cell, disabling its normal function.

Stem cell

Relatively unspecialized cell that can reproduce indefinitely and differentiate into specialized cells; can reproduce itself indefinitely.

Gene tracking technology used in order to compare amount of mRNA in several samples @ one time.

Reverse transcriptase - Polymerase Chain Reaction

RNA sequencing

State of the art gene tracking technology used to synthesize and sequence cDNA in order to look at all mRNAs in a cell. -cut shorter and converted into cDNA

Methylation of histone tails in chromatin can promote condensation of the chromatin. (T O F)

T

Which of the following functions are characteristic of general transcription factors in eukaryotes?

They bind to other proteins or to the TATA box.

gene therapy

alteration of an afflicted individual's genes

Enyzmes are usually composed of _____(A)______. But Primase is an enzyme composed of _____(B)______.

amino acids, nucleotides

Transgenic (Pharm) animal

an animal that has genes from another species of animal.

miRNAs can control gene expression by what action? See Concept 18.3 (Page)

binding to mRNAs and degrading them or blocking their translation

Which of the following processes would result from a mutation that deactivates a regulatory gene of a repressible operon in an E. coli cell?

continuous transcription of the structural gene controlled by that regulator Submit

cDNA

copy DNA - made using reverse transcriptase - a copy of DNA made from RNA to then be used in amplification processes

Griffith found:

first to reveal the "transforming principle," which led to the discovery that DNA acts as the carrier of genetic information In his work with pneumonia-causing bacteria and mice, Griffith found that some CHEMICAL from the smooth cells was TRANSFERRED to rough cells, transforming them into smooth cells

What process repairs damage to a preexisting double helix?

nucleotide excision repair nuclease cuts out and replaces damaged stretches of DNA

Chargaff's rule

percentages of bases:21.5 A 21.5 T 28.5 C 28.5 G

Among the newly discovered small noncoding RNAs, one type helps to reestablish methylation patterns during gamete formation and blocks expression of some transposons. Which of the following types of RNA is responsible for this function?

piRNA

The action of helicase creates _____.

replication forks and replication bubbles *A replication fork is the transition region between paired and unpaired DNA strands.

Which of these is(are) pyrimidines?

C, D, and E *Pyrimidines are single-ring structures

Biotechnology

The manipulation of organisms or their components to make useful products.

Polymerase Chain Reaction

The most common/FASTEST method used to obtain copy of specific DNA gene -heatshock, cold ice, cacl2

Reproductive cloning via nuclear transplantation process

- A cell is taken from a donor(1) and de differentiated. - An egg is taken from another donor(2) and the nucleus is removed. - The cell and egg are fused and the nucleus from donor(1) becomes the egg's nucleus. - the egg is grown in culture into an embryo and into the uterus of a third donor.

Reason why cloned individuals don't always look/behave identically

- Because of the random deactivation of the X chromosome in females. - Epigenetic changes - Heterozygous at a particular allele.

Major potential uses for iPS cells

- Cells from patients with diseases can be reprogrammed and be model cells for studying the disease/treatments - Could be used to replace nonfunctional tissues in patients.

Next generation sequencing/ Sequencing by Synthesis process

- DNA is fragmented, isolated and isolated with a bead. - Fragments are amplified (10^6) and attached to the bead at the 5' end. These are placed into a well with primers and DNA polymerases. - A solution of all four nucleotides is added to the wells and washed off. - There is a flash if a nucleotide is joined to the DNA strand from pyrophosphate being released. The patterns of the flashes determines the sequence.

Why eukaryotic genes are difficult to express in bacteria

- Differences in control sequences and promoters. - The presence of introns, no RNA-splicing machinery.

How to determine a gene's function

- Disable the gene and observe the consequences - Data about time of gene expression and its location. - A gene sequence can be compared to other species where its function is known, could indicate a comparable function.

Genome-wide association studies process

- Groups of people with the same genetic condition are analyzed to find nucleotide changes specific to the condition. - Tests for genetic markers that are different amongst individuals.

DNA Microarray Assays process

- Isolate the mRNAs made in a cell of interest and use them to make cDNA. - the cDNA is made with fluorescent molecules and allowed to hybridize to the DNA array. - cDNAs from different tissues are usually labeled with differently colored fluorescent molecules and tested on the same microchip. - The cDNA is attached to a glass slide in an array.

Challenges associated with animal cloning technology

- Many cloned animals have defects. - A small percentage of cloned embryos develop normally to birth. - Many epigenetic changes must be reversed in the nucleus of a donor animal for gene expression programs to work properly.

Benefits to using eukaryotic cells to express eukaryotic genes.

- Some eukaryotes (yeast) also have plasmids. - They can modify proteins after translation that would not function otherwise (glycolipids etc.)

Why Bacterial plasmids are used as cloning vectors.

- They are easy to obtain, be manipulated, and introduced into bacterial cells. - They can reproduce quickly.

Gene cloning.

- To make copies of a particular gene for study. - To produce a protein product. A type of DNA cloning producing multiple copies of a single gene. never live as long because epigenetics messes it up

Reverse Transcriptase - Polymerase Chain Reaction process (RT-PCR)

- cDNA is synthesized. - PCR is carried out. - Gel electrophoresis reveals the amplified DNA products in samples that have the mRNA transcribed from the specific gene.

How restriction enzymes are used during DNA cloning to make a recombinant DNA plasmid.

- restriction enzyme cuts the sugar-phosphate backbones. - DNA fragment from another source cut by the same enzyme is added. - DNA ligase seals the strands.

In a nucleotide, the nitrogenous base is attached to the sugar's _____ carbon and the phosphate group is attached to the sugar's _____ carbon.

1'-5'

cloning genes in bacterial plasmids (five steps)

1. obtain engineered plasmid DNA and DNA from hummingbird cells. They hummingbird DNA contains the gene of interest 2. cut both DNA samples with the same restriction enzyme, one that makes a single cut within the lacZ gene and many cuts within the hummingbird DNA 3. mix the cut plasmids and DNA fragments. Some join by base pairing; add DNA ligase to seal them together. The products include recombinant plasmids and may non-recombinant plasmids 4. mix the DNA with bacterial cells that have a mutation in their own lacZ gene. Some cells take up a recombinant plasmid or other DNA molecule by transformation 5. plate the bacteria on nutrient containing agar medium supplemented with ampicillin and x-gal, a molecule resembling lactose. Incubate until colonies grow RESULTS: only a cell that took up a plasmid, which has the amp gene, will reproduce and form a colony

Plasmid

A small, circular DNA molecule replicated separately in bacteria.

gel electrophoresis

A technique that uses gel to separate a mixture of nucleic acids or proteins based on size, charge, or other physical properties. DNA is negative

in vitro mutagenesis

A technique used to disable a gene in order to observe its function in a cell/organism.

What are two problems with bacterial gene expression systems, and how is each solved?

Getting a cloned eukaryotic gene to function in bacterial host cells can be difficult because certain aspects of gene expression are different in eukaryotes and bacteria. To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active bacterial promoter just upstream of the restriction site where the eukaryotic gene can be inserted into the correct reading frame. Another PROBLEM with expressing cloned eukaryotic genes in bacteria is the presence of noncoding regions (introns) in most eukaryotic gene very long and unwieldy, and they prevent correct expression of the gene by bacterial cells, which do not have RNA-splicing machinery. This problem can be surmounted by using a cDNA form of the gene, which includes only the exons.

Reproductive cloning "Dolly"

Organismal cloning in which the goal is to make another individual.

Short Tandem Repeat(STRs)

Tandemly repeated units of 2-5 nucleotides in a specific part of the genome. - PCR is used to amplify particular STRs - gel electrophoresis is used to determine the length of the region and the number of repeats.

Nucleic Acid Hybridization

The base pairing of one strand of nucleic acid to the complementary sequence on a strand from another nucleic acid molecule through hydrogen bonds.

Double-stranded viral DNA is incorporated into a host cell as a _____.

provirus "Provirus" is the name given to double-stranded viral DNA that has been incorporated into a host cell's genome. Return to Assignment

SSB proteins

single stranded binding proteins that help keep the DNA uncoiled

You can tell that this is an image of a DNA nucleotide and not an RNA nucleotide because you see a _____.

sugar with two, and not three, oxygen atoms

Which of the following conditions is most likely to cause the lactose operon to be transcribed?

the cyclic AMP and lactose levels are both high within the cell

Nuclear transplantation

the nucleus of a un/fertilized egg is removed and replaced with the nucleus of a differentiated cell.

describe how short tandem repeats can produce a sensitive genetic profile

the number of repeats present inthese regions is highly variable from person to person, and for one individual, the two alleles of an STR may even differ from each other

Sticky end

the single stranded end on the double-stranded restriction fragment that base pairs with another complementary sticky end. -form hydrogen bonds with complementary sticky ends, make permanent with DNA ligase -ex: used in recombinant plasmids