Mol bio 2

Restriction Enzymes

Enzymes that cleave DNA into fragments at or near specific recognition sites within molecules known as restriction sites.

DNA Ligase

An enzyme that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond.

Reverse Transcriptase

An enzyme that is an RNA dependent DNA polymerase (RdDp) that catalyzes the production of complementary DNA or cDNA from an RNA template.

Taq Polymerase

Thermostable Polymerase enzyme from Thermophilus aquaticus (Taq).

Melting temperature (Tm)

Temperature at which 50% of a DNA fragment is denatured, dependent on DNA length and GC content.

Thermocycler

Instrument used in PCR that automates the repeated cycles of heating and cooling.

PCR

"Polymerase Chain Reaction"; an in vitro method for amplifying a specific DNA segment that hybridize s opposite ends of the segment in opposite polarity, over repetitive cycles exponentially replicate that particular segment

Nested PCR

PCR that uses primers that are within the DNA sequence that has been previously amplified by another set of primers.

Multiplex PCR

multiple PCR products of distinct regions and sizes are PCRed simultaneously.

Short Tandem Repeats (STR)

Accordion-like stretches of DNA containing core repeat units of between two and seven nucleotides in length that are tandemly repeated half dozen to multiple dozen times

5' UTR

The exonic region that is after the start of transcription but before the start of translation.

3' UTR

The exonic region that is after the end of translation but before the end of transcription.

Oligonucleotide

Chemically synthesized small nucleotide polymers usually shorter than 50 nt and synthesized from 3'>5'.

Poly-A tail

A polynucleotide tail found at the 3' end of all RNA transcripts made from RNA polymerase II. These RNAs are called mRNAs

Oligo-dT

A primer used to synthesize cDNA's using hybridization to a terminal structure found at the 3' end of all mature mRNAs.

cDNA

A duplex DNA with a strand identical to the mRNA of a specific gene; made by reverse transcriptase.

cDNA library

Clones of DNA in Vectors List Every Transcript (CDVL-ET RT → cDNA Library Every Transcript) resulting in the transcriptome of that sample.

Consensus Sequence

A DNA or amino acid sequence consisting of the most common residues to occur at a given site in a set of similar sequences.

Alternative Splicing

gene transcripts can be spliced differently to produce distinct (diff) RNAs that code for distinct proteins.

5' methyl-cap

A residue of 7-methylguanosine linked to the 5'most residue of an mRNA through a unique 5',5' triphosphate bond.

Exons

Regions of transcripts exported from the nucleus that contain the protein coding domains as well as UTRs.

snRNP's ('snurps')

Ribonucleoprotein complexes formed by proteins that bind small nuclear RNAs (snRNA) found in the nucleus, forming parts in spliceosome

Branch point

An internal A residue upstream of the 3' splice site of an intron that binds the U2 snRNP and attacks the 5' splice site forming the loop of the intron lariat

Intronic Splice Silencer (ISS)

Cis elements in intronic regions that have domains that shut down splicing to proximal exons.

Intronic Splice Enhancers (ISE)

cis elements in intronic regions that have domains that recruit splicing to proximal exons.

Exonic Splice Silencer (ESS)

cis elements in exonic regions that have domains that shut down splicing in proximal exons.

Exonic Splice Enhancers (ESE)

cis elements in exonic regions that have domains that recruit splicing to proximal exons.

Exon Definition

The process in which a pair of splicing sites are recognized by recruitment of splicesome machinery components around an alternative exon. It is the sum of interactions of splice enhancers and silencers to specify inclusion of an exon in exported mature mRNA.

Introns

Regions of transcripts that are removed before export from nucleus to make mature mRNA.

Isoforms (splicing or gene)

any of two or more functionally similar sequences of nucleic acids or proteins that have a similar but not identical sequences that arise from interchanging parts.

Exon Shuffling

The hypothesis credited to W. Glibert that genes have evolved by the recombination of various exons coding for functional domains.

Unequal Crossing-over

Results of errors in pairing and crossing over between non-equivalent sites in the genome. It results in regions of duplications and deletions on recombinant chromosomes.

GFP (Green Fluorescent Protein)

The first visual marker developed from a jellyfish protein for observing living eukaryotic cells; frequently used marker for promoter activity in cells.

CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats DNA breaks; a technique used to edit genes with single base pair resolution and is more efficient than double recombinant homologous recombination method.

sgRNA

is a short synthetic RNA composed of a scaffold sequence necessary for Cas-9 binding and a region of about 20 nucleotides for specific binding.

tracrRNA

• T: tracrRNA

• A: Attached to guide RNA

• C: Constant base stretch

• S: Stem-loop structure

TACoS

Is the part of the sgRNA known as the trans-activating crRNA. It is made up of long stretch of bases that are constant and provide the stem loop structure bound by CRISPR nuclease. To generate specificity for a residue, it is attached to a specific guide RNA.

PAM (protospacer adjacent matrix)

is a DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in CRISPR. PAM is a requisite component for cutting, but is not included in the guide RNA.

Transgene

is the piece of DNA from any source that is used to generate an organism modified with its addition.

Transposon

Transposable elements are cassettes of DNA that are able to insert themselves (or a copy) at a new location in the genome without having any relationship with the target locus.

gRNA

is a short synthetic RNA composed of a scaffold sequence necessary for Cas-9 binding.

Autonomous transposon

transposons that can synthesize transposase and all the proteins necessary for mobility and the sites recognized by these enzymes.

Non-autonomous transposon

Contain the sites for recognition but lack the enzymes necessary for transposition. Those are supplied from transposons elsewhere in the genome.

Conservative transposon

A transposon that precisely excises and inserts into a new site. No growth in numbers of transposons.

Replicative transposon

A transposon that replicates to copy and insert into a new site. Leaves one copy in the original position and one in a new site. At least 2 copies per event.

SINES

Short interspersed nuclear element; are non-autonomous Alu repeat containing retrotransposons.

LINES

Long interspersed nuclear element; are autonomous Alu repeat containing retrotransposons.

Inverse PCR

Allows amplification of unknown sequences flanking a known insertion sequence.

Which of the following enzymes is used in Molecular Biology to join two strands of DNA being Spliced together

DNA Polymerase

Taq Polymerase

Reverse Transcriptase

Restriction Enzymes

DNA Ligase

DNA ligase

Which of the following enzymes Is used in Molecular Biology to cut strands of DNA at a recognition site that is normallv palindromic: sometimes, these are called the scissors of the cell

DNA Polymerase

Tag Polvmerase

ever Transcrintase

Restriction Enzymes

DNA Ligase

Restriction enzymes

3. Which one of these characteristics is NOT a property of a plasmid - an episomal DNA fragment used to carry and replicate our favorite DNA fragments in vivo?

a.Selectable marker for maintenance of the plasmid - such as antibiotic resistance.

b. A Green Fluorescent Protein gene - to select transformations that glow

C. Origin of Replication - to replicate many copies of the plasmid in the bacteria

d. Multiple Cloning Site - to make it easy to insert copies of our gene into the

b

In the plasmid prep that we did in the lab (boiling prep) or shown in the book (alkaline lysis), after you

break open the bacteria and precipitate all the junk (bacterial cell wall, proteins and genomic DNA), where do you find the plasmids?:

a. In the pellet

b. In the supernatant

b

What does the dyad symmetry in restriction enzyme cleavage relate to in terms of DNA -protein interaction

a. That the protein wraps the DNA like a pig in a blanket

b. That there is dyad symmetry in binding two proteins in opposite directions on the DNA

b

Using the NEB 1kb ladder to the right, you can see that the marker goes from 10kb at

the top to 0.5kb at the bottom with regulars size markers distributed along the way.

Assuming DNA is negatively charged, and the DNA is run through an agarose gel with

a current running through it, what lead would you expect at the bottom of the gel

Positive lead

Negative lead

Positive

What size is the doublet used for orienting the marker in the gel?

1 kb

2kb

3kb

Thick band 3

Which of the pieces of the plasmid are used to select the plasmid on selective plates and grow the plasmid in bacteria on selective media?

The purple region that is depicted as mini-white. This is a fly eye-color gene

The yellow region depicted as ori. This is the so-called origin of replication where the replication

fork begins in this plasmid for copying DNA.

The orange and purple region depicted with the restriction enzymes, Not I, Xho I and Xbal and EcoRI

that might loosely be called a Multiple Cloning Site (MCS)

The green arrow depicted as Amp', suggesting that it gives resistance to the antibiotic Ampicillin.

d

If you cut this plasmid, where would you expect the fragment that contains the GFP responsive piece and cut with Xba I and Not I to run in an Agarose gel. In other words, which fragment would contain the orange and purple piece.

a. 484 base pairs

b. 7844 base pairs

a. the orange and purple piece containing the "insert" described is 484 bps

10. Here is the digest of the plasmid you isolated (for some students) run on a gel. Where is the 484 base pair

fragment? Image shows gel with may and dark bands on top near neg, is a. B is on positive/bttm end light few bands

b

Using the markers next to the gel pictured here, make your guess as to which band

IS larger than SUV base pairs.

The top band above the .5 kb mark