MIDTERM LEC: ANALYSIS & CHARACTERIZATION OF NUCLEIC ACIDS & PROTEINS

Question 1

Set of techniques & methods used to study & understand the structure, function, interactions, & characteristics of nucleic acids & proteins in biological systems

Answer 1

NUCLEIC ACID/PROTEIN ANALYSIS

Question 2

what are the methods for NUCLEIC ACID/PROTEIN ANALYSIS

Answer 2

A. Restriction Enzyme Mapping of DNA
B. CRISPR Enzyme Systems
C. Hybridization Technologies

Question 3

• Involves restriction enzymes (endonucleases): type II is commonly used, with 4-6 bp recognition/restriction sites, or binding/cutting sites on DNA
▪ Used to identify the position of restriction sites in a DNA fragment in relation to each other
▪ Developed using small circular bacterial plasmids

Answer 3

RESTRICTION ENZYME MAPPING OF DNA

Question 4

RESTRICTION ENZYME MAPPING OF DNA what type is commonly used???

Answer 4

type 2

Question 5

Developed using small circular bacterial plasmids

Answer 5

RESTRICTION ENZYME MAPPING OF DNA

Question 6

Restriction enzyme digestion of DNA fragment/region = pattern of fragments

Answer 6

Used to identify that DNA

Question 7

▪ Resulting differences in the size/number of restriction fragments
▪ Useful in epidemiological studies
▪ The basis of the 1st molecular-based
human ID & mapping methods
▪ Clinical analysis of structural changes in chromosomes associated with disease

Answer 7

Restriction fragment length
polymorphisms (RFLPs)

Question 8

RESTRICTION MAPPING USES/APPLICATIONS??

Answer 8

▪ Gives an idea of genome sequence
▪ Used to map genomes when sequence information is unknown
▪ Can be used to introduce primers for sequencing
▪ Used for ID & characterization of naturally-occurring plasmids & to engineer the construction of recombinant plasmids

Question 9

▪ Found in archaea & bacteria
▪ Guides a common enzyme to specific sites determined by RNA components
▪ Useful for manipulation of both DNA sequence & RNA expression

Answer 9

CRISPR ENZYME SYSTEMS

Question 10

CRISPRs MEANING?

Answer 10

CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS

Question 11

▪ Protective system using the invading DNA to target itself
▪ Encodes an endonuclease: CRISPR-associated protein (Cas)

Answer 11

CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPRs)

Question 12

CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPRs) Encodes an endonuclease:

Answer 12

CRISPR-associated protein (Cas)

Question 13

CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS

Types:
Type 1 and 2 targets???

Answer 13

dsDNA

Question 14

CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS

Types:
Type 3 targets??

Answer 14

ssDNA & RNA

Question 15

Hybridization reaction that is used to analyze the nucleic acid content of an unknown sample

Answer 15

HYBRIDIZATION TECHNOLOGIES

Question 16

formation of H bonds between 2 complementary strands of nucleic acids

Answer 16

Hybridization

Question 17

In hybridization technologies: 1 strand is unlabeled & 1 strand is labeled (with isotopes/ fluorescence) = _____

Answer 17

probes

Question 18

▪ ss fragment of nucleic acid(NA)/protein with a detectable signal that specifically binds to complementary sequences/ target protein
▪ Purpose: identify 1 or more sequences of interest within a large amount of NA

Answer 18

PROBES

Question 19

southern blot target???

Answer 19

DNA

Question 20

northern blot target???

Answer 20

RNA

Question 21

Western blot target??

Answer 21

Protein

Question 22

south western blot target???

Answer 22

protein

Question 23

eastern blot target???

Answer 23

protein

Question 24

far eastern blot target???

Answer 24

lipids

Question 25

southern blot probe???

Answer 25

nucleic acid

Question 26

Northern blot probe???

Answer 26

nucleic acid

Question 27

western blot probe??

Answer 27

protein

Question 28

South western blot probe???

Answer 28

DNA

Question 29

eastern blot probe??

Answer 29

Protein

Question 30

far easter blot probe??

Answer 30

none

Question 31

target NA has to contain the sequence of interest

Answer 31

In order the probe to bind

Question 32

Denaturation of dsDNA
probes before use:

Answer 32

❖ Heating the probe (95ºC, 10-15 min) in hybridization solution
❖ Treating with 50% formamide/2x SSC at a lower temperature for a shorter time (75ºC, 5-6 min)

Question 33

▪ Early methods: Fragment of gene was cloned on a bacterial plasmid 🡪 isolated by restriction enzyme digestion & gel purification 🡪 labeling & denaturation of fragment 🡪 applied to Southern/Northern blot
▪Isolation of sequence of interest from viral genomes
In vitro organic synthesis of a predetermined sequence 🡪 only for short, oligometric probes
▪ Synthesized using PCR

Answer 33

DNA PROBE

Question 34

RNA PROBES
Source: transcription from a synthetic DNA template in vitro
▪ Predesigned systems commercially available
▪ Southern blot: coding strand transcript
▪ Northern blot: antisense transcript ▪Labeled with a radioactive/modified
nucleotide for production of signal

Answer 34

RNA PROBE

Question 35

OTHER NA PROBE TYPES

Synthetic & resistant to nucleases:

Answer 35

peptide NA probes, locked NA probes, unlocked NA probes

Question 36

products of a generalized response to a specific antigen

Answer 36

Polyclonal antibodies