Lab 8 DNA Fingerprinting

What are the 2 types of bases?

Purines and Pyrimidines

What way does DNA run?

Antiparallel

What are the purines?

Adenine and guanine

What are the pyrimidines?

Cytosine, thyamine, uracil

What structure contains cytosine?

DNA and RNA

What containes thymine?

DNA

What contains uracil?

RNA

What do hydrogen bonds link?

Bases on opposite strands

How many bonds between A and T?

2

How many bonds between C and G?

3

What are phosphodiester bonds for?

Link 5’ phosphate and 3’ hydroxyl

What do phosphodiester bonds form?

DNA backbone

Where are the sugar phosphates located?

Outside of helix

Where are the bases of DNA located?

Interior

Restriction buffers?

NaCl, Tris-HCl, Mg2+

What are the 2 restriction enzymes?

EcoRI and PS+1

What overhang does EcoRI have?

5’

What overhang does Pst1 have?

3’ overhang

DNA restriction enzymes

Defense evolved by bacteria to protect against viral infection

How do restriction enzymes work?

enzymes destroy DNA from invading bacteriophages

Restriction endonucleases

Sit along helix and when recognizing the specific sequences they cut or chemically separate the DNA molecule at the site

Restriction site

The specific sequence/site where the enzyme cuts or digests the restriction site

How many base pair sequences do enzymes recognize?

4 or 6

What are the 2 restriction enzymes?

EcoRI and PS+1

What is the temperature you incubate the enzyme?

37 degrees Celsius

What happens at too hot temperatures?

Enzymes denature (killed)

What happens at too cold temperatures?

Enzyme activity lowered = longer digestion time

Multiple fragments

Multiple restriction sites causes enzyme to cut at each and every of those sites

How many fragments do circular fragments have?

# of fragments is same as number of restriction sites

How many fragments do linear have?

fragment number is equal to number of restriction sites +1

NaCl

Provides correct ionic strength

Tris-HCl

Provides proper pH(0.8)

Mg 2+ (magnesium)

Enzyme cofactor

What does DNA fingerprinting require?

Polymerase chain reaction (PCR) and Restriction fragment length polymorphism (RFLP)

Is PCR done in lab?

No

What does PCR do?

Amplify/increase the number of DNA copies in a sample

What do primers do?

Ensure specificity- designed to be complementary and anneal to specific regions on each side of DNA target region

How are PCR highly specific?

Only produces copies of a desired sequence from sample DNA

Restriction Fragment Length Polymorphism

Exploits variations (DNA nucleotide mutations) between individuals

What causes difference in number and size of fragments?

Some sites are cleaved and recognized by enzymes while others are not.

RFLP

Differences and variations among DNA sequences at sites recognizes by restriction enxymes

Agarose

Large pores/matrix which helps separate DNA fragments

Buffer

Allows the current to flow through the gel

Where is buffer placed?

Within the apparatus

Sample loading dye

Helps visualize the sample and track the movement of bonds; does not stain DNA itself

How can DNA fragments be visualized by?

Fast blast DNA stain and SYBR/greenglo safe DNA dye

When are gels stained in fast blast?

After electrophoresis

When are gels dyed with SYBR

Before casting/ before electrophoresis