Lecture 10: DNA at High Resolution:The Use of DNA Cloning, PCR, Hybridization and Gene Expression

I. Importance

II. Recombinant DNA Technology

III. Restriction enzymes (Cutting the DNA: restriction enzymes serve as molecular scissors)

A. Restriction enzymes fragment the genome at specific sites

B. Different restriction enzymes produce fragments of different lengths

1. The size of the recognition site is the primary determinant of fragment length

2. The timing of exposure to a restriction enzyme helps determine fragment size

C. Different restriction enzymes produce different numbers of fragments from the same genome

IV. Molecular Cloning.

Purification and amplification of fragments for storage and analysis

Cloning step 1: ligation of fragments to cloning vectors creates recombinant DNA molecules

1. Sticky ends facilitate recombinant DNA fabrication

2. There are several types of vectors

A. Plasmid Vectors

B. Phage and cosmid vectors

C. YAC vectors

D. Identifying Transformants

1. Transformation: vectors carry insert DNA into cells

2. How do you know which cells have been transformed?

3. How do you know whether the plasmids inside those cells contain an insert?

4. Amplification of the recombinant DNA molecule: the actual cloning

E. Purifying Cloned DNA

Cloned DNA is purified by various means that separate recombinant plasmid from host

DNA, then insert from vector

1. Purifying recombinant plasmid from host DNA

2. Purifying inserts from plasmids

Libraries are collections of cloned fragments

1. Genomic libraries are random collections of the DNA fragments from the chromosomes

of a given species, inserted in a suitable vector

F. CDNA cloning

cDNA libraries carry information from the RNA transcripts present in a particular tissue

G.. Genomic versus cDNA libraries

V. Southern Blots: Identifying and isolating clones of interest

A. Screening with DNA probes: hybridization to complementary sequences picks out fragments

of interest

1. What is a DNA probe?

2. How to obtain a DNA probe

3. Screening with DNA probes

4. Requirements and limitations of hybridization probes

B. Screening through expression: genes cloned in specialized vectors produce proteins that light

up with specific antibodies

VI. The polymerase chain reaction (PCR) can amplify small DNA fragments of partially known

sequence for further analysis

1. How PCR achieves the exponential accumulation of target DNA

2. PCR products can be analyzed and utilized just like cloned restriction fragments

3. PCR has many uses

VII. DNA Sequencing

VIII. Gene Expression

IX. Terms and Concepts to Know: PCR, cloning, automated DNA sequencing, partial digest, different vectors, cosmid, phage, YAC, plasmid, recognizing transformation and inserted fragment, genomic library, cDNA library, reverse transcriptase, screening with probes, gene expression and microarrays

Figures and Tables in Chapter 9: 2-6, Table 2, 7-11, 13, 15-18, Chapter 10: 24 and 25