Molecular Genetics

Question 1

What is DNA fragmentation and how does it occur?

Answer 1

Using restriction enzymes DNA is cut into smaller pieces, they cut the sugar-phosphate backbone and cap it off

Question 2

Restriction enzymes are:

Answer 2

very specific and each will cut a particular 4-6bp sequence typically

Question 3

What are sticky ends?

Answer 3

stick ends means that the overhang wants to bind to a complementary sequence if after the original cut it is left alone it will reform

Question 4

How can sticky ends be used?

Answer 4

Fragment DNA add digested DNA from another source it can be ligated with the original DNA to create an intentional insertion (ligation only occurs with identical sticky ends)

Question 5

How is a southern blot performed?

Answer 5

1. DNA digested (restriction enzymes)
2. separation of DNA fragments by size using gel electrophoresis
3. transfer of fragments to a nitrocellulose or nylon membrane
4. hybridization of a probe to the fragment. or fragments of interest
5. probe detection

Question 6

What information can you gain from Southern Blots?

Answer 6

tell you both the size and something about the sequence of a fragment mixed in with many other fragments

Question 7

Southern Blot uses:

Answer 7

RFLPs- mutations changed sequence size, genetic disease identification, genetic markers in gene mapping, VNTRs highly polymorphic (variable) used for genetic fingerprinting

Question 8

What is DNA Cloning?

Answer 8

capture fragment of DNA in a lab organism and culture to produce large amounts of the same piece of DNA for further manipulation

Question 9

What are libraries of clones

Answer 9

collection of clones, each containing different DNA fragment

Question 10

Steps of DNA cloning:

Answer 10

1. RE digestion, vector DNA digested to have identical sticky ends, mixed with ligaments
   2.certain bacteria take up pieces of DNA (competent bacteria) live cells take up recombinant vector process of bacteria taking up new DNA is called transformation

Question 11

What are plasmid vectors?

Answer 11

circular DNA found in bacteria does not contain any bacterial chromosomal DNA, has origin of replication and RE sites to see if cloning worked

Question 12

How to further amplify DNA?

Answer 12

pick colony to grow, toothpick colony, remove liquid, lose cells, extract DNA from lipids proteins and RNA

Question 13

Is it possible to generate a collection of all expressed genes?

Answer 13

cDNA libraries: cDNA is complementary DNA

Question 14

What is PCR?

Answer 14

Polymerase Chain Reaction is an exponential amplification of a piece of DNA without a live cell

Question 15

PCR steps:

Answer 15

1. Develop primers complementary to sequences flanking target DNA
2. add DNA with primers free nucleotides and polymerase
3. heat DNA to unwind double helix
4. lower temp allow primers to bind
5. polymerase ,ales complementary strands of the now separate strands
6. repeat

Question 16

What are microarrays?

Answer 16

arrayed sequences of thousands of microscopic spots of DNA oligonucleotides each containing pic moles of a specific DNA sequence (probes)

Question 17

How can you make microarrays?

Answer 17

Photolithography, Spotting of probes, printing of probes

Question 18

What are Affymetrix arrays?

Answer 18

arrays consist of 25mer oligonucleotide probes where every perfect match probe has a mismatch probe - allows for SNP detection

Question 19

What is Sanger sequencing?

Answer 19

sequencing by termination, targeted

Question 20

What is NGS?

Answer 20

sequencing by synthesis, illumina sequencing, random fragmentation, coverage

Question 21

New Genome Sequencing tech:

Answer 21

Direct DNA sequencing without amplification, nanopore technology, silicone chip technology

Question 22

How does illumina sequencing work?

Answer 22

relies on the attachment of randomly fragmented genomic DNA to a planar optically transparent surface, amplified, templates sequences, high sensitivity fluorescence