MOLBIO - MT 122 - Lecture 9 - Restriction Endonucleases and RFLP Typing

Deoxyribose

Backbone sugar of DNA is?

Hydrophobic

Bases are described as ___ which is why the sugar phosphate backbone needs to twist around each other protecting the bases.

1. DNA replication
2. DNA transcription
3. DNA translation

What comprises the central dogma of molecular biology?

d. Both statements are incorrect.

Helicase - splits helical strands
DNA polymerase - Elongates strand

In replication, the helical structure is split into two by DNA polymerase. While the helicase elongates the strand, comprising the leading and lagging strand.

a. First statement is correct. Second statement is incorrect.
b. First statement is incorrect. Second statement is correct.
c. Both statements are correct.
d. Both statements are incorrect.

Exonuclease

A nuclease that degrade DNA from free 3'OH or 5'-phosphate end.

Endonuclease

A nuclease that cleave DNA internally by cutting the phosphodiester backbone.

c. Both statements are correct

Remember: Exo requires ends

Endonucleases makes internal cuts. Exonucleases removes nucleotides from the ends.

a. First statement is correct. Second statement is incorrect.
b. First statement is incorrect. Second statement is correct.
c. Both statements are correct.
d. Both statements are incorrect.

Restriction Endonucleases

Remember: they recognize specific base sequences and break or restrict the DNA polymer at the sugar-phosphate backbone.

These are also known as DNA scissors.

Type 3 restriction enzyme

Name the restriction enzyme:

-Both nucleases and methylase activity
-Cleavage of DNA occurs at 24-26bp from site to 3' side

Type 1 restriction enzyme

Name the restriction enzyme:

-Both nucleases and methylase activity
-Bind to host specific sites of 4-6bp
-Contain methylated adenine

Type 2 restriction enzyme

Name the restriction enzyme:

-Nuclease function, no methylation activation
-Bind to symmetrical 4-8bp DNA recognition sites

Type IV restriction enzyme

Name the restriction enzyme:

-Both nuclease and methylase activity
- Asymmetrical cut sites

a. First statement is correct. Second statement is incorrect.

It has palindromic sites (when you read it forward and backward, it says the same).

Type 2 restriction enzyme is the most commonly used in the laboratory. It has asymmetrical cut sites.

a. First statement is correct. Second statement is incorrect.
b. First statement is incorrect. Second statement is correct.
c. Both statements are correct.
d. Both statements are incorrect.

"Sticky ends"

Nature of this particular arrangement is when it anneals or when it sees a structure complementary to it, it would directly anneal or attach to it.

a. First statement is correct. Second statement is incorrect.

Sticky ends must have MATCHING overhangs.

Blunt ends have no overhangs and are formed by different enzymes that can be joined together, regardless of recognition site. Sticky ends also has no overhangs but recombination is only at specific recognition sites.

a. First statement is correct. Second statement is incorrect.
b. First statement is incorrect. Second statement is correct.
c. Both statements are correct.
d. Both statements are incorrect.

Restriction Mapping

Knowing the location of a specific restriction type in a DNA strand or sequence is called?

TRUE

TRUE OR FALSE

Restriction Site Mapping was developed using circular plasmids.

DNA ladder

Serves as a reference and allows us to map/know the size of the DNA sequences.

Double-digestion

Putting two restriction enzymes in the same reaction or tube is called as?

Restriction Fragment Length Polymorphism

The resulting differences in the location and number of restriction sites is called?

● Genetic Mapping
● Parentage Testing
● Human Identification
● DNA Fingerprinting

What are the common applications in RFLP?

Long arm of chromosome 17 in the BRCA1 gene

What chromosome was linked to inherited breast cancer founded by Dr. Mary Claire King?

STAR activity

Remember: The higher the star activity, the more specific the restriction enzyme cuts the DNA sequence.

Refers to the altered specificity of restriction enzymes

Shortened reaction time and inorganic solvents

(Prolonged reaction time and organic solvents like ethanol are preferred)

Which of the following conditions does not favor STAR activity?

-Suboptimal buffer
-High concentrations of enzyme
-Cation Imbalance
-Alkaline pH
-Shortened reaction time
-Inorganic solvents

Southern Blot

DNA fingerprinting was first introduced by what method of Sir Alec Jeffrey's?

b. First statement is incorrect. Second statement is correct.

1-20 kb size DNA are required

Requirements of southern blot include 50-500 kb size of DNA. 100ng to 1ug of high quality DNA are also needed.

a. First statement is correct. Second statement is incorrect.
b. First statement is incorrect. Second statement is correct.
c. Both statements are correct.
d. Both statements are incorrect.

5-7 days

32P-based probe system would yield results in how many days?

TRUE

TRUE OR FALSE

Under nonstandard conditions, restriction enzymes bind and cut sequences that are unspecified or not desirable.

d. Both statements are incorrect.

Nucleoside has a nitrogenous base + pentose sugar + phosphate group, Nucleotide has a nitrogenous base +
pentose sugar.

a. First statement is correct. Second statement is incorrect.
b. First statement is incorrect. Second statement is correct.
c. Both statements are correct.
d. Both statements are incorrect.

FALSE

Single-locus probe (SLP) is commonly used nowadays since MLPs would result in highly variable resultant patterns and actually required expertise to optimize and interpret.

TRUE OR FALSE

Multiple-locus probes (MLPs) are more commonly used in DNA fingerprinting.