Recombinant DNA & Biotechnology

How is DNA manipulated?

• isolate DNA (from…) - properties of chromosomal DNA (large, acidic) - properties of vector DNA

• digest DNA - shearing (intentional, unintentional) - restriction enzymes

• recognition sequences = specific (Why?)• palindromic: same sequence antiparallel

GAATTC

CTTAAG

What is recombinant DNA?• DNA molecule made in the laboratory derived from at

least two genetic sources.

Lecture 9 – Recombinant DNA

How is DNA manipulated?

• staggered vs blunt (sticky or cohesive)

Lecture 9 – Recombinant DNA

• directed vs undirected

EcoRI BamH1

EcoRI BamH1

BamH1 BamH1

BamH1 BamH1

BamH1 BamH1

EcoRI BamH1

Lecture 9 – Recombinant DNA

How is DNA manipulated? • isolate DNA (from…) - properties of chromosomal DNA (large, acidic) - properties of vector DNA

• clone fragments into vector - attributes

1. ori2. mcs/polylinker3. selection

a. vectorb. insert

4. small size5. high copy #6. host range7. size of insert8. Growth9. Purification10. Stability11. Storage12. expression

Vectors – Plasmids, YACS, cosmids, TI plasmid – T DNA, viruses

Lecture 9 – Recombinant DNA

Identifying recombinant plasmids• transformation/selection/transgenic

Lecture 9 – Recombinant DNA

Useful Markers• selectable• screenable

Libraries

Lecture 9 – Recombinant DNA

- noncoding

Libraries

Genomic cDNA

- what cells, tissue, organ, developmental stage?

- abundance

- how to clone

Oligo dT

Reverse transcriptase

DNA pol

RNaseH

- digestion partial vs complete

- size/representation: # of clones x insert size = 10X genome size

- stability repetitive DNA

• mRNA (how do we isolate??)1. location? (cytoplasm)2. processing? (poly A tail)

• isolate DNA (from…)- genomic

Lecture 9 – Recombinant DNA

Identifying recombinant plasmids

Large

Small

• characterize fragments - gel electrophoresis (DNA properties) - restriction mapping

transform

Identify correct plasmid

Lecture 9 – Recombinant DNA

Identifying recombinant plasmids

Complementarity!!!- Southern Blot- Northern Blot- sequence analysis

How do we identify DNA/RNA clone of interest??

- Run DNA/RNA- transfer to filter- hybridize probe to filter- detect signal

Lecture 9 – Recombinant DNA

Southern analysis:

RI RI RI

5kb 12.5kb

1

5kb

12.5kb

2 1

12.5kb

2

probe

DNA GelSouthern blot

Lecture 9 – Recombinant DNA

Northern:- isolate mRNA (from…)- electrophoresis (ssRNA vs dsDNA –

structure)

- transfer of mRNA to filter (wicking)- incubate w/ labeled DNA (ss probe)- wash- develop (radioactive vs

nonradioactive)

Northern analysis:

1

5kb

12.5kb

2

probe

Region of genome is transcribed =

How do we identify DNA/RNA clone of interest?

gene!

Lecture 7 – Recombinant DNA

Gene analysis on a global scale - microarrays

1. Array DNA sequences – oligonucleotides (what defines a gene)

3. Label samples – distinct fluorescent dyes

4. Hybridize to microarray

5. Detect fluorescent signals – expression ratio

2. Isolate mRNA - distinct samples

Yellow:Red:Green:

DNA manipulation – hybridization…

Lecture 9 – Recombinant DNA

oncogenicwild type

Lecture 9 – Recombinant DNA

Gene analysis on a global scale - microarrays

6. Uses ?

- temporal changes in gene expression during development- different tissues- +/- drug, hormone etc…- Wild type vs disease (define diagnostic profiles)- Genotyping

Lecture 9 – Recombinant DNA