TRM Week 3 - Transcriptomics

omics refers to

a field of study in Biological Sciences that ends with -omics and it has a prefix such as genomics, transcriptomics, proteomics, or metabolomics

genetic engineering refers to

the use of "in vitro" techniques to alter genetic material in the laboratory that may be reinserted into the original source organism or into some other host organism

genetic engineering depends upon

our ability to cut DNA into specific fragments and to purify these fragments for further manipulation

basic techniques of genetic engineering?

restriction enzymes

gel electrophoresis

nucleic acid hybridization

nucleic acid probes

molecular cloning

cloning vectors

What do restriction enzymes do?

also called restriction endonucleases, recognize specific DNA sequences and cut DNA at those sites; they're essential for in vitro DNA manipulation in genetic engineering

What were restriction enzymes' original job inside prokaryotes?

widely spread among prokaryotes (rare among eukaryotes)

they were the prokaryotes immune system. they protected prokaryotes from hostile foreign DNA while also protecting their own DNA from inadvertent destruction

what are the three major classes of restriction enzymes?

Type I, Type III, and Type II

what is the difference between the major classes of restriction enzymes?

Type I and type III bind to the DNA at their recognition sequences but cut a considerable distance away.

In comparison, Type II cleaves the DNA within their recognition sequences, which makes them more useful for specific manipulation of DNA.

Type II are always dimers.

what type of restriction enzyme is always used for genetic engineering?

Type II since its more useful for specific DNA manipulation since it cuts the DNA within their recognition sequence

Every restriction enzyme has its own

recognition sequence! also in order to add fragment DNA into the cut DNA you need to cut the fragment DNA and the cut DNA you want to put it in with the same restriction enzyme in order for the sticky ends to match and be ligated together

What is the restriction enzyme from E. Coli?

EcoRI; can also be modified by EcoRI methylase which protect restriction site from being cut by EcoRI

what type of recognition sequences do the restriction enzymes bind to/recognize?

inverted repeat sequences (Palindromes such as anna, atta, cggc) typically theyre 4-8 base pairs long

what is the EcoRI recognition sequence?

6 base pair sequence, GAA/TTC (cut between the A and T)

most restriction enzymes are composed of

two identical polypeptide subunits. each subunit recognizes and cuts the DNA on one of its two strands, a double stranded beak. leaves short single stranded overhands called Sticky Ends, or Blunt Ends.

What are modification enzymes?

modification enzymes protect cell's DNA from inadvertent destruction by restriction enzymes. each restriction enzyme is partnered with a corresponding modification enzyme that shares the same recognition sequence

how do modification enzymes protect the cell's DNA from restriction enzymes?

they recognize the same recognition sequence as their corresponding restriction enzyme

they chemically modify nucleotides in restriction recognition sequence

usually Methylation of DNA (adding a methyl group to the DNA makes it unreadable to the restriction enzyme, therefore stopping the read and the cutting of the DNA)

what is EcoRI's modification enzyme called?

EcoRV

what does gel electrophoresis do?

gives visualization of the size difference, separates DNA molecules based on size

how does a gel electropheresis work?

electrophoresis uses an electrical field to separate charged molecules.

the DNA/nucleic acids are negatively charged due to their phosphate groups and will move towards the positive electrode side. (NEG TO POS)

gels are usually made of 1.5 to 2% agarose gel

what can you stain gel electrophoresis with in order to be visualized under UV light?

ethidium bromide, DNA fragments can then be purified and use for different purposes

what will happen to the same DNA that is cut by different restriction enzymes?

the same DNA cut by different restriction enzymes will have different banding patterns on an agarose gel, the size of fragments can be determined by comparision to a standard

what is a restriction map?

a map of the location of restriction enzyme cuts on a segment of DNA, also called optical mapping and it has been used to create restriction maps of the chromosomes of E coli and thei bacteria

What is nucleic acid hybridization?

when DNA is denatured, the single strands can form hybrid double stranded molecule with other single stranded DNA or RNA by complementary base pairing.

so one DNA and one RNA hybrid double helix.

widely used in detecting, characterizing, and identifying segments of DNA

what are nucleic acid probes?

segments of single stranded nucleic acids whose identity is already known and used in hybridization. to allow for detection, probes can be made radioactive or labeled with chemicals

what can hybridization be useful for?

can be very useful for finding related sequences in different chromosomes or to find the location of a specific gene

what is the target fragments of SOUTHERN BLOTTING?

probes of known sequences are hybridized to target DNA fragments that have been separated by gel electrophoresis. probe can be DNA or RNA

what is the target fragments of NORTHERN BLOTTING?

probes of known sequences are hybridized to target RNA fragments that have been separated by gel electrophoresis. probe can be DNA or RNA

what is the target fragments of WESTERN BLOTTING?

probes of known sequences are hybridized to target PROTEIN that have been separated by gel electrophoresis. probe can be DNA or RNA

What is the S. Blotting procedure?

left panel contains purified DNA from several plasmids treated by restriction enzymes then run through gel electrophoresis

right panel contains southern blot

aft blotting, DNA in the gel is hybridized to a radioactive probe and the bands are visualized with X ray autoradiography

notice that if the lane contains the DNA marker, none will be complementary to the labeled probe and no bands will hybridize

what is the transcriptome?

can be defined as the complete set of RNA being produced from the genome at a given point in time

the advancement of RNA based sequencing technologies has allowed for what?

allows for the determination of both the presence and amount of different RNA molecules

Conventional or "traditional' PCR is:

QUALITATIVE, NOT quantitative. aka traditional PCR just says if it is there or not.

what are the steps in PCR (polymerase chain reaction)?

1. Template DNA is DENATURED by heating (hottest temperature 94 degree C)

2. Two artifical DNA primers flanking the target DNA (ANNEAL 55 degree C) are present in excess

3. DNA polymerase EXTENDS (72 degree C) the primers using the original DNA as the template.

PCR is also called

DNA Amplification.

PCR does not copy whole DNA molecules but amplifies stretches of up to a few thousand base pairs (the target) from within a larger DNA molecule (the template). turns one copy into two in one run, then two copies into four copies, four into eight, then so on and so forth

what type of DNA polymerase must be used in PCR and why?

Taq polymerase (from bacteria) or Pfu polymerase (from archaea) because they can handle the temperatures that PCR must be done in. Human polymerase would denature in these high temperatures

what are some applications of PCR?

PCR is a powerful tool that is extremely sensitive, specific, and highly efficient.

• phylogenetic studies to amplify genes from various sources

• surveying different groups of environmental organisms

• amplifying small amounts of DNA like from mummies or fossilized plants and animals

• identifying a specific bacteria

• looking for a specific gene

• forensics to identify human individuals from very small samples of their DNA

what is qPCR?

quantitative PCR, also called real-time PCR or RT-qPCR, can be used to determine the quantity of individual transcripts in a cell. used in Gene Expression Analysis (HOW MUCH of a gene is being expressed)

what are the steps in qPCR?

• messenger RNA is isolated to assess the "purity" of RNA

• then converted to cDNA through reverse transcription

• then the cDNA is used as the target DNA in a PCR reaction carried out in the presence of a dye that fluoresces when bound to the duplex

RT- qPCR used for gene expression analysis

What are the applications of qPCR?

gene expression

determination/monitoring viral load

quantifying cancer genes

microarray verification

transgenic copy

SNP - predict response to drugs, toxins, disease, etc.

Microarrays are

used in transcriptomics

also known as gene chips, small solid-state supports to which genes or portions of genes are fixed and arrayed spatially in a known pattern

gene segments come from PCR

better to use cDNA than mRNA because it is too unstable

How do you make a microarray?

From short single-stranded oligonucleotides corresponding to all the genes of an organism synthesized and affixed at known locations

gene chip is assayed by hybridizing fluorescently labeled mRNA from cells grown under a specific condition to the DNA probes, then later scan the chip with a laser

how do you know a gene has been transcribed on the microarray/gene chip?

hybridization between a specific mRNA and a DNA segment on the chip indicates that the gene has been transcribed

what does color represent in DNA microarrays?

the color represents the relative expression level of the genes. Red represents HIGH expression while green represents LOW expression

what can be learned from microarray experiments?

expression of specific groups of genes under different conditions

What are the applications of microarrays?

to study the extent to which certain genes are turned on or off (RNA is isolated and measured)

used in clinical diagnostic tests to see if mutations are present

used to determine how genes may cause body to respond to drugs

may be replaced by sequencing sometimes, but still relevant