MMG 115 Midterm One

What keeps the two DNA strands together?

Hydrogen bonding and hydrophobic interactions (base-stacking)

Purines

A and G

Pyrimidines

C and T

How many hydrogen bonds do G and C base pairs have?

Three (stronger than A and T)

How many bonds do A and T share?

Two

What does it mean when DNA replication is semi conservative?

each daughter strand contains one parental strand and one new strand

Primase

The starting point for DNA polymerase, makes primer

DNA polymerase

adds nucleotides complimentary to the template

Helicase

opens the DNA helix, breaking hydrogen bonds

SSB (single stranded DNA-binding protein)

binds to ssDNA at the helix fork to prevent re-annealing

topoisomerase

relaxes DNA from supercoil

DNA ligase

joins the 3’ Oh and 5’ phosphate of adjacent DNA and okazaki fragments

How many DNA polymerases does E. coli have?

2, DNA pol l (removes primers and fills okazaki fragment gaps) and DNA pol lll (replication of the genome, very large and complex)

DNA polymerase common features

uses ss template, require DNA/RNA primer with 3’ OH to start, polymerize new strand 5’ to 3’

Features of E. coli Pol l

DNA dependent DNA pol

5’ to 3’ exonuclease activity

5’ to 3’ polymerase activity

3’ to 5’ exonuclease activity

5’ to 3’ polymerase activity

requires DNA template, DNA/RNA primer, 3’ OH, Mg++, dNTPs

5’ to 3’ Exonuclease activity

needs dsDNA and MG++

is on during 5’ to 3’ polymerase activity (coupling this is needed for the Nick Translation Reaction)

removes primers

can be separated from polymerase activity by protease cleave or genetic engineering in order to make Klenow fragments

Nick Translation Reaction

nick DNA with DNase l, then translated 5’ to 3’, used to label DNA fragment with fluorescent/radioactive dNTP labels

3’ to 5’ exonuclease activity

used for proofreading, removes incorrect nucleotides

blocked by polymerase activity

trims 3’ overhangs

polymerase activity balances nuclease = blunt ends

T4 DNA Pol

DNA dependent DNA polymerase

5’ to 3’ pol activity

no 5’ to 3’ exonuclease activity

strong 3’ to 5’ exonuclease activity

Reverse Transcriptase

RNA dependent DNA polymerase

ss RNA/DNA templates

polymerizes 5’ to 3’

needs DNA/RNA primer

no exonuxlease activity

Primer Annealing

primer finding the target sequence

temperature changes : denatured and renatured

Nucleic Acid Hybridization

temperature increases, breaking bonds and creating ss nucleotides

random collisions of ss nucleic acids with complementary sequences, helix nucleation, rapid zippering

if incorrect base paring occurs they are unstable and don’t last long

for a new double helix to form both strands must be open and unfolded (free of secondary structures, so this is done at high temperatures or in the presence of organic solvent

During Tm how much of the DNA is ds and ss

½ for both

perfect conditions for hybridization

increased temperature, increased concentration of solvent, decreased concentration of salt, increased temperature of detergent

what happens when hybridization is in less stringent conditions

usually there are mismatches, but sometimes stable hybrids will form (used as a strategy to experimentally introduce mutations)

Conventional PCR

amplification of DNA sequences between two primers

Components of PCR

DNA template, 2 ss oligonucleotide primers, DNA polymerase, dNTPs, buffer conditions

What is Taq DNA polymerase?

thermo-stable DNA dependent DNA polymerase

removes the need for enzymes needed for DNA replication at the replication fork

5’ to 3’ polymerase activity

5’ to 3’ exonuclease activity when 5’ to 3’ polymerase activity is on

no 3’ to 5’ exonuclease activity

Proteins required for E coli in vivo DNA replication

DNA polymerase, primase, helicase, SSB, topoisomerase, DNA ligase

Proteins required for PCR

primers are added, DNA polymerase, heat takes care of most of the other things

Quantitative PCR

detects the amplification target at end point (plateau stage of PCR amplification)

What is the difference between conventional and quantitative PCR

conventional: end point measurement, detects after PCR, can’t tell the starting amount of DNA sequence

Q-PCR: continuous measurement, you can see when the reaction enters the exponential phase and the plateau

SYBR green dye

dye binds to the minor grove of DNA, fluoresces under blue light and increases brightness after binding to dsDNA

SYBR green based QPCR

plot fluorescence against PCR cycle numbers

there are three stages: fluoresence does not bind above the detection baseline, rise above baseline (increase exponentially, amplification levels off

Pros and Cons of SYBR green based QPCR

pros: cost effective, easy to design

cons: reaction specificity based on primers, SYBR green can also bind to primer dimers and pcr products from mis annealed primers

Dissociation curve

performed at the end of the PCR reaction (assesses non-specfic products)

there are three steps

1)all products from PCR amplification are melted

2) the temperature decreases for complete annealing of dsDNA

3) The temperature of the PCR mix is gradually increased where the fluorescence intensity is monitored and recorded for the curve (different products have different melting points impurities will create a different peak then desired products)

What does a single peak mean on a dissociation curve graph

pure single amplicon

What does more than one peak mean on a dissociation curve graph

multiple amplicons present

TaqMan Probe based QPCR

a probe is included in the reaction with a reporter fluorescence dye on the 5’ end and a quencher on the 3’ end

5’ to 3’ exonuclease activity chews away at the sequence with the probe during extension

then

fluorescence when the fluorescence is released into the solution and is far from the quencher

Pros and Cons of TaqMan Probe based QPCR

pros: very specific, multiple reactions can happen in one tube, the probe can be labelled with different fluorescence

cons: it is expensive and harder to optimize

What is the CT sequence

the number of cycles required for any one cycle to reach the threshold of fluorescence

threshold is sent in the exponential phase of amplification plot

value is inversely proportional to the log of the initial target concentration

what does a low ct value mean

higher concentration of initial target sequence

what was the restriction of phage growth due to in bacteria

specific sequence endonuclease cleavage, makes ds breaks at or near recognition sites, cuts up foreign DNA

How do the bacterial cells protect their own DNA

hemi-methylated sequences by restriction methylase, freshly replicated host DNA is methylated to protect against cleavage

Type one restriction enzymes

needs s-adenosyl methionine and ATP to propel DNA translocation through the enzyme molecule, cuts DNA 1-5 kb away from the recognition sequence, methylase and endonuclease are a part of the same complex

Type three restriction enzymes

uses ATP to move along the DNA, cuts 24-28 bps from one side of the recognition site, methylase and endonuclease are one complex

Type two restriction enzyme

uses Mg++, (4,6,8 bp long with two fold axis of rotational symmetry , defined site close or in recognition sequence, methylase and endonuclease are two separate complexes

Calculate the average fragment size made by restriction enzyme

4^ #bp recognition

What are the different ends created by restriction enzymes

blunt ends and sticky ends (5’ overhang and 3’ overhang)

isoschizomers

different enzymes that recognize the same site and cleave at the same site

neoschizomers

different enzymes that recognize the same site but don’t cleave the same site

dam methylase

methylates A in GATC sequences, inhibit cleavage

dcm methylase

methylates the c in CCA/TGG sequences

what is the efficiency of restriction digestion affected by

supercoil of dna, nature of flanking dna, Mg++

DNA ligase

require proximal 3’ OH and 5’ P groups

don’t fill in gaps

don’t work on ss nucleic acids

T4 DNA ligase

uses ATP for energy, ligate nicked DNA, blunt ends, and sticky ends

works in three steps

1) enzyme adenylation

2)Transfer of AMP

3)nucleophillic attack

E. coli DNA ligase

uses NAD for energy

ligate nicked DNA and complimentary stick ends (not blunt ends)

alkaline phosphotase

removes terminal monophosphates from DNA, RNA, and dNTPs, does not remove phosphate from within the phosphodiester bonds

T4 Polynucleotide Kinase

catalyzes the transfer of phosphates on polynucleotides, preps DNA fragments for ligation, helps with radio labelling DNA fragments

s1 nuclease

ss specific endonuclease, degrades ss nucleic acids and any ds with ss because of nicked,gap, mismatch, or loop

RNase H

endonuclease with little sequence specificity, degrade RNA in a duplex with complimentary strand of DNA (hybrid), does not degrade ss/ds DNA or RNA

DNase l

DNA specific endonuclease with little sequence specificity, in presence of Mg++ it nicks each strand of dsDNA independently in random fashion, in presence of Mn++ it cleaves both strands of dsDNA at the same site and makes the same breaks

T5 Exonuclease

dsDNA specific exonuclease and ssDNA endonuclease, cleaves linear/nicked dsDNA in the 5’ to 3’ direction, not active on full circular or supercoiled DNA

general structure of gene

5’ promoter, TSS, RBS, translation start site, orf, translation stop site, terminator, 3’