Recombinant DNA Basics #1

Question 1

What is recombinant DNA?

Answer 1

any DNA molecule formed by joining DNA segments from different sources

Question 2

How are staggered or “sticky” ends made on DNA?

Answer 2

restriction endonucleases cleave DNA at specific internal sequences

Question 3

Describe how restriction enzymes and DNA ligase cut and paste DNA

Answer 3

2 DNA pieces with the same unique restriction site on each end can be digested with restriction enzymes then mixed and treated with DNA ligase to give joined fragments

Question 4

Describe the 2 most common ways of visualizing recombinant DNA

Answer 4

1. Agarose - large fragments (>500 nucleotides), horizontal gel, most used
2. Polyacrylamide gel electrophoresis (PAGE) - small DNA fragments (<500 nucleotides)

Question 5

What are the contributing factors to the amazing specificity of DNA annealing?

Answer 5

strand length (longer = more stable)
pH, salt, temp (high pH, salt, and temp = destabilizing)
sequence (G-C stronger than A-T)

Question 6

How can DNA annealing be used to find a target gene?

Answer 6

combining a mixture of single stranded DNA probes for gene A & single stranded DNA molecules

Augmenting the temperature of the mixture will yielf different stringencies of DNA helices that formed. higher temps have increased stringencies and lower temp will have decreased stringencies resulting in the potential for imperfect base pairing

This allows the detection of complimentary sequences

Question 7

Describe how PCR works

Answer 7

PCR allows amplification of any gene via base pairing specificity

the cycle begins with your target sequence. Primers are added that attach to the target sequence’s ends and then the sequence is duplicated each cycle

Question 8

Describe the main components of an operon

Answer 8

regulatory gene - codes for a protein that binds DNA and influences transcription of the structural gene
operator - DNA sequence that binds the regulatory protein
promotor - DNA sequence than binds RNA polymerase in absence of repressor
structural gene - codes for non-regulatory proteins that have activity in metabolism

Question 9

Describe the lac Operon in E. COli

Answer 9

Encodes 3 structural genes (lac Z, lacY, and lacA) and 1 regulatory one (lacI)
lac i - encodes the lac repressor, a protein that binds an operator sequence upstream of the structural genes and blocks transcription