5 - Manipulation of Nucleic acids

Question 1

nucleases

Answer 1

enzymes that degrade nucleic acid. RNases and DNases. most are specific, varying degree of specificity. som attack only ss, others only ds, some both.
exonucleases attack ends of nucleic acids, only remove one or short piece of ssDNA. Either 3’ or 5’, not both.

Endonucleases cleace the chain in the middle. Some nonspecific, some (like REs) extremely specific.

Question 2

how do REs distinguish between self-DNA and foreign DNA?

Answer 2

modoification enzymes bind to the same restriction sites as REs and add a methyl group to the DNA, usually to A od C. In a few cases it is sulfur in stead of methylation.

Question 3

Recognition of DNA by REs

Answer 3

recognition sites are usually 4, 6, or 8 bases long, inverted repeats.

Question 4

isoschizomers

Answer 4

REs from different species with the same recognition site. Do not necessarily cut the same place in the sequence.

Question 5

Type I REs

Answer 5

cut DNA 1000 or more bp away from rec site. loops the DNA around so the enzyme binds both the rec site and cut site.

One subunit binds to rec site, another methylates it, third cuts at a distance away. exact distance varies -> not of practical use to mol bio. suicidal (inactivated after one cut).

Question 6

Type II REs

Answer 6

cut in rec site. of use in genetic engineering as exact cut-site is known. can leave blunt or sticky ends. Sticky are most useful.

If two sticky ends made by the same enzyme is ligated, it may be cut again. If the ends are made by different enzymes, a hybrid is formed and can not be cut by the same enzyme.

Star activity might be a problem

as opposed to Type I, the modification protein and RE are separate proteins.

Question 7

star activity

Answer 7

REs may cut the DNA at slightly different sites than their rec sites in suboptimal conditions. can also occur if excess enzyme per DNA, or if reaction incubates too long. generally avoided in lab by specific buffers that provide optimal conditions.

Question 8

DNA fragments are joined by DNA ligase

Answer 8

DNA ligase cobvalently links the fragments of the lagging strand during DNA replication. Will ligate two fragments touching end-to-end.

difficult to ligate blunt ends - slow and requires high concentration of ligase and DNA. bacterial ligase cannot join blunt ends at all, but T4 can.

Question 9

Making a restriction map

Answer 9

restriction map = diagram that shows where REs cut on a segment of DNA.
made by seq the DNA and scanning for restr sites.

Question 10

restriction fragment lenght polymorphisms (RFLP)

Answer 10

related molecules of DNA, such as different versions of the same gene from two related organisms, normally have very similar seq, and thus similar restriction maps, but differences in one base can make it so that it is no longer a restricrtion site.

If two such related but different DNA molecules are cut with the same RE, segments of different lengths are produced, resulting in different band sizes on a gel. A difference between two DNA segments that affects a restriction site is known as a RFLP, and may be used to identify organisms or analyze relationships even if the function of the altered segement is unknown.

Question 11

How to chemically synthetize DNA

Answer 11

must anchor the first nucleotide to a solid support, usually controlled pore glass (CPG) are used (beads that have pores in uniform sizes). The beads are organized in a column and the reagents are poured down the column. nucleotides are added one by one and the growing strand of DNA remains attached to the glass beads until the synthesis is complete.

1) first base is anchored to bead via 3’ OH. Added with no phosphate groups. Bound to bead with scaper molecule (to prevent other bases from interacting with the bead.
2) acid (often trichloroacetic acid, TCA) is poured through the column, removing DMT blocking group and exposing the 5’ OH on the first nt. Second nt added, linking to the first via the single phosphate in the phosphoradmidite moiety. Column is washed with acetonitrile and argon between each step. acetonitrile removes any inreacted reagents, argon removes any excess acetonitrile.

Question 12

Problems for chemical DNA synthesis

Answer 12

1) each deoxynucleotide has two OH groups, one for bonding to the nucleotide in front and one for the one behind. Chemicla reagents cannot tell the difference between these, so one has to be blocked while the other is activated. This is done by phosphoreamidite method. Proceeds in 3’->5’. before adding a new nucleotide, the 5’ OH of the previous nucleotide is blocked with DIMETHOXYTIL (DMT) group, and the 3’ is activated with a phosphoreamidite. Note that the reagents for chemical synthesis are phosphoamidite nucleotides (not nucleoside triphosphates), and that the synthesis commences in 3’->5’ direction (opposite of biological synthesis).

Question 13

Chemical synthesis of a complete gene

Answer 13

Sequences up to 80 or 100 nt can be synthetized in one segment, longer have to be made in different segments and combined.

A series of overlapping fragments, representing both strands of the gene to be assembled, are manufactured. These are purified and annealed together.

2 alternatives:
1) synthetize whole gene with only nicks between, and ligate them

2) synthetize smaller fragments that overlap on each end enough to make the two strands stay together, leaving large ss-gaps that DNA polymerase I can fill in before the ligation.

Question 14

PNA

Answer 14

peptide nuleic acid, polypeptide backbone with NA bases as side chains.

totally artificial molecule

DNA analog in genetic engineering

PNA-strand can bind to complementary strand of RNA and DNA.

uncharged backbone

really fucking bad at penetrating cells = major clinical problem. Could possibly be carried by positively chrged liposomes.

Question 15

PNA clamp

Answer 15

two identical PNA strands that are joined by a flexible linker and are intended to form a triple helix with a complementary strand of DNA or RNA

Question 16

Nucleic acid mimics

Answer 16

PNA

LNAs (locked nucleic acids)

morpholine nucleic acids

can form duplexes and triplexes, greater stability than natureal nucleic acids.

LNA in hybridization assays that need high specificity between target and the probe.

morpholino nucleic acids substitute a morphino ring for deoxyribose, and are cheaper to make than natural DNA, extremely resistant to nuclease degradation, highly soluble.

Question 17

Measuring DNA and RNA concentration with UV

Answer 17

DNA and RNA absorb UV light at maximum 260 nm.

Like a regular spectrometry thing.

Proteins absob at 280 nm. The relative purity of DNA can be found by the 260/280 nm absorbtion ratio. For pure DNA the A260/A280 ratio is 1,8. Pure RNA has 2,0.

Question 18

Radioactive labeling of nucleic acids

Answer 18

radioisotope = radioactive form of an element.

DNA = “hot” if radioactive, “cold” if not.

Most important radioactive forms of elements in molbio: 32 P and 35 S.

Since S is not usually in DNA/RNA, we use phosphorothioate derivatives of the nts, in which one of the four O-atoms around the chemical phosphorus is replaced by a sulfur.

Question 19

Detection of radioactive DNA

Answer 19

2 methods;

liquid: scintillation counting
flat: autoradiography

scintillation counting:
relies on special chemicals called scintillants, which emit a flash of light when they absorb high-energy electrons, known as beta-particles, which are released by certain radioactive isotopes, including 32P and 35S. the ligh pulses are detected by a photocell. a scintillation counter detects and counts the light pulses.

autoreadiography:
gels containting DNA/RNA are transferred to membraned by blotting, or gel can be dried onto filter paper. The radioactive DNA/RNA will emit beta particles, which turns regular photographic film black. Just lay one on top of the thing and wait a few hours (in a dark room).

Question 20

Fluorescence in the detection of DNA and RNA

Answer 20

fluorescence = process in which a molecule absorbs light of one wavelength and then emits light of another, longer, lower energy wavelength.

need a beam of light to excite the dye, and a photo-detector. different dyes have different wavelengths.

FACS = fluorescent activated cell sorter
instrument that sorts cells (or chromosomes) based on fluorescent labelling.

Question 21

chemical tagging with biotin or digoxigenin

Answer 21

biotin = vitamin
digoxigenin = steroid from foxglove plants

both linked to uracil, so to tag DNA uracil must be incorporated. UTP is modified to deoxyuridine triphosphate (deoxyUTP) labeled with biotin/digoxigenin, and added to a DNA synthesis reaction. DNA polymerase will insert the labeled uridine instead of thymidine. biotin/digoxigenin sticks out without disrupting the structure of DNA.

detection of biotin/digoxigenin:
2 steps:

1) use a molecule that recognizes biotin/digoxigenin.
biotin:
since biotin = vitamine, needed by animals and many bacteria, chicken eggs (bacteria paradise due to nutrients) need to be protected. Avidin binds biotin so avidly that bacteria that try to infect the egg become vitamin deficient.

digoxigenin:
uses Ab

Ab and avidin can be visualized easily in many ways. Option 1 is to attach an enzyme that generates a colored product to the avidin/Ab.

avidin can be conjugated to alkaline phosphatase, which produces a blue dye when cleaving the artificial chromogenic substrate X-phos.

another option for biotin/avidin or digoxigenin/Ab detection is chemiluminescence, in which an enzyme that produces light by a chemical reaction is used. Alkaline phosphatase is still linked to avidin/Ab, and lumi-phos is added. Cleaving leads to light.

Question 22

electron microscope

Answer 22

materials that absorb the electrons appear darker.

Question 23

Hybridization of DNA and RNA

Answer 23

Hybrid DNA = artificial dsDNA molecule made by pairing two single strands from different sources. By adding both to the same container, increasing the temp to break the H-bonds between the ds, re-annealing can happen between two strands of different origin, as long as they are (nearly) complementary. To avoid reannealing immediately, the sample can be attached to a filter.

hybridization:
formation of dsDNA molecule by annealing two single strands from two different sources.

Can be used to study relatedness between two DNA molecules (hemoglobin-gene, f.ex), or isolate genes for cloning

Question 24

Southern, Northern and Western blotting

Answer 24

Southern: used to identify corresponding genes in different organisms. One DNA sample (target) is hybridizes to another (probe). only hybridization of DNA to DNA.

Suppose we have a large DNA molecule (yeast) and wish to locate a gene whose sequence is similar to the human gene of interest.

1) target (yeast) DNA is cut with RE
2) fragments separated by gel electrophoresis
3) ds fragments are melted into ss fragments by soaking the gel in alkaline denaturing solurtion (such as sodium hydroxide).
4) DNA fragments are transferred to a nylon membrane
5) membrane is dipped in a solution of labaled DNA probe molecules (like a radioactive segment of the human gene). The probe binds only to thise fragments with similar seq.
6) a piece of photographic film is placed on th emembrane and a black spot appears corresponding to the hybrid molecule.

Northern:
hybridization with RNA as target molecule and DNA as probe. DNA probes may be used to locate mRNA that correspond to the same gene. the mixture of RNA is run on a gel and transferred to a membrane, which is then probed as in Southern.

Western has to do with proteins, and comes in chap 15

Question 25

Zoo blotting

Answer 25

not distinct method, but neat trick in Southern blotting.
most of higher animals DNA is noncoding, but we want to analyze the coding parts. Noncoding DNA mutates rapidly, and change quickly, whereas the coding regions change more slowly, and can still be recognized after years of divergence between two species.

DNA is extracted from a series of related species. Samples in this DNA “zoo” are cut with RE, and fragments are run on a gel and transferred to a nylon membrane. They are probed using DNA that is suspected of bein human-coding DNA. If a DNA sample really does include a coding sequence, it will probably hybridize with some fragment of DNA from most other closely related animals. If noncoding; probably only hybridize to the human dna.

Question 26

FISH

Answer 26

fluorescence in Situ Hybridization

In the other techniques the nucleic acid has to be isolated.
FISH detects the presence of a gene (or corresponding mRNA) within the cell. DNA seq from the GOI must be generated for use as a probe. these may be obtained by cloning the gene, or, more usually nowadays, amplified by PCR. The whole gene seq is rarely needed as a probe, unless it is essential to distinguish between closely related genes. As the name indicates, the probe DNA is labaled with a fluorescent dye (can be localized under fluorescence microscope).
The tissue or cell must also be trated to denature the target DNA; but this is done on the actual tissue, leaving the DNA in its original location. If RNA is the target the cells to not ahve to be treated, as its ss.

Used for checking for hte presence of the same or similar genes in related organisms, or in chimeric organisms created by genetic engineering

Probe does not need to represent coding DNA and FISH can be used to search for any known DNA seq, including repetitive DNA or transposable elemnts, etc. Also check if viral DNA is in cytoplasm, f.eks. Can also be used to localize a gene on a chromosome in metaphase.