Chapter 20-21: DNA tools and biotechnology

DNA technology

methods for analyzing/manipulating DNA

Genetic engineering

direct manipulation of genes

Nucleic Acid hybridization

base pairing between complementary strands

recombinant DNA

DNA from multiple sources combined

cloning vector

DNA molecule that carries foreign DNA (e.g) plasmid

Plasmid

a small circular bacterial DNA molecule

Sanger sequencing

• uses dideoxynucleosides (ddNTP’s)

• these terminate DNA synthesis (no 3’OH group)

• produces fragments of different lengths

how to read sequencing gel

read BOTTOM to TOP

shortest fragments travel farthest

each band = nucleotide

gives a sequence of newly synthesized strand

Plasmid role

• replicate independently

• carry inserted foreign DNA

• used as cloning vectors

why are plasmids useful

easy to isolate

easy to insert DNA

replicate quickly in bacteria

Gene cloning

1. insert gene into plasmid

2. put plasmid into bacteria

3. bacteria replicate → copy gene

4. produces many identucal DNA copies

How recombinant DNA is made

1. restriction enzymes

   1. cut DNA at specific sequences (restriction sites)

   2. Often produces sticky ends

2. sticky ends

   1. single-stranded overhangs → base pair with matching DNA

3. DNA ligase

   1. seals DNA fragments together

GEL ELECTROPHORESIS principle

DNA is placed in a gel + electrical field

DNA moves towards the positive electrode

smaller fragment moves farther/faster

GEL ELECTROPHORESIS interpretation

• bottom bands = smaller fragments

• top bands = larger fragments

• used to compare sizes and verify recombinant plasmids

PCR (Polymerase Chain Reaction) steps

1. Denaturation

   1. dna strands separate

2. Annealing (cool)

   1. Primers bind

3. Extension

   1. DNA polymerases builds new strands

PCR key componets

primers- define region to copy

TAQ polymerase- heat-stable enzyme

PCR importance

• can amplify from

  • tiny samples

  • crime scenes

  • ancient DNA

Genomics

study of entire genome

Bioinformatics

computational analysis of DNA data

Human Genome Project: Map- based approach

Genome mapped first

then sequenced in order

Human genome Project Whole-genome shotgun (venter)

• DNA is cut into random fragments

• each fragments sequenced

• computer assembled full sequence