Chapter 10

Question 1

Human Genome Project

Answer 1

Create an accurate sequence of the human genome

Question 2

Restriction Enzymes

Answer 2

Recognizes a specific sequence of bases anywhere on the genome
- restriction fragments generated by digestion of DNA

Question 3

Restriction Enzyme: Fragment Ends

Answer 3

Blunt ends - cuts are straight through both DNA strands at line of symmetry
Sticky ends - cuts are displaced equally on either side of the line of symmetry

Question 4

Restriction Enzymes: Fragment Lengths

Answer 4

4-base restriction size - 256 bp
6-base recognition - 4100bp

Question 5

Mechanical Forces

Answer 5

Used to fragment DNA at random locations
Can break phosphodiester bonds

Question 6

Gel Electrophoresis

Answer 6

Distinguishes DNA fragments by size
Movement of charged molecules in an electric field
- Visualize DNA fragments by staining gel with fluroescent dye

Question 7

Molecular Cloning

Answer 7

Purifies specific DNA fragment from other segments and makes identical copies

Question 8

Molecular Cloning: Step 1

Answer 8

Creating recombinant DNA molecules with plasmid vectors:
Plasmid cloning vectors - have origin of replicators, selectable marker, synthetic polylinker
Digestion - of vector with restriction enzymes males complementary sticky ends
DNA ligase - seals the phosphodiester backbones between vector and inserted fragment

Question 9

Molecular Cloning: Step 2

Answer 9

Host cell takes up and amplifies recombinant DNA
Transformation - process by which a cell takes up foreign DNA
only cells with plasmid will grow on media with ampicillin - produce colony on agar

Question 10

Genomic Library

Answer 10

long-lived collection of cellular clones that contain copies of every sequence in the while genome
each colony has different recombinant plasmid - each with a part of the human genome

Question 11

DNA Polymerase Requirements:

Answer 11

Template - single strand of DNA to copy
Deoxyribonucleotide triphosphate - building block for new DNA
Primer - free 3’ end to which DNA polymerase can attach new nucleotides

Question 12

Sanger Sequences

Answer 12

Uses DNA polymerase to make new DNA
Recombinant plasmid is a good template
generates a series of single-stranded DNA fragments

Question 13

Automated DNA Sequencing

Answer 13

Fragments flow past a laser beam and color of terminal base is digitally recorded

Question 14

Hierarchical Strategy

Answer 14

1. construct BAC genomic library
2. identify sets of overlapping BAC clones
3. Shear DNA fro, each BAC separately to make similar clones
4. sequence DNA
5. Assemble sequences based on overlap
   Strategy was successful, labor-intensive, inefficient and expensive