HRR: tools of molecular genetics I and II Flashcards
Describe restriction endonucleases and how they cleave DNA at specific sequences
They allow for enzymatic digestion of DNA at a specific sequence, often a palindrome. They often create overhangs, either 3’ or 5’
How does gel electrophoresis separate DNA fragments?
By size; smaller fragments move farther in the gel toward the positive end, while larger fragments stay closer to the negative end
Explain how restriction endonucleases and DNA ligases are used to create recombinant DNA
The cut DNA fragments can be ligated together to form recombinant DNA. Overhangs that are complementary with other fragments cut from the same enzyme can anneal. They must be complementary, usually meaning they’re cut with the same enzyme!!
What is the purpose of Southern Blotting?
detect specific DNA fragments
Describe the process of Southern Blotting
-DNA is digested into fragments by restriction endonucleases
-DNA fragments are separated by size via gel electrophoresis
-DNA is denatured into single strands and transferred to a positively charged nylon membrane
-A radioactive probe complementary to a specific sequence in a DNA fragment is used to preform hybridization
-Detect the probe that hybridized to the fragment, and observe the fragment
What is the purpose of northern blotting?
it detects specific RNAs
Describe the process of northern blotting
-Extract RNA from cells or tissue
-Separate RNA molecules by size via gel electrophoresis
-Transfer RNA molecules to a positively charged membrane
-A radioactive probe complementary to a specific sequence in an RNA molecule is used to preform hybridization
What is the purpose of western blotting
detects specific proteins
Describe the process of western blotting
-Separate proteins by size via gel electrophoresis
-Transfer to membrane and incubate with an antibody to detect protein
Briefly describe PCR
DNA amplification via taq polymerase; uses sequence specific primers to amplify target DNA sequences. It can also be used with mRNA, with an additional step of using a reverse transcriptase
Specify the 3 main steps of a PCR cycle in amplification of DNA target sequences.
-Denaturation of DNA into single strands via heat
-Annealing of sequence specific primers to complementary DNA sequences in target DNA
-Synthesis of target DNA molecules via taq polymerase
Describe the technique of methylation-sensitive PCR.
It can be used to differentiate between methylated and unmethylated alleles.
- DNA samples are treated with bisulfite.
-The bisulfite will convert unmethylated cytosines to uracil, and methylated cytosines remain unchanged as they are resistant to chemical modification by bisulfite.
What kinds of primers are needed in methylation-sensitive PCR?
Two different primers will be needed, as in unmethylated there is a base change!
What are genomic libraries?
Comprised of all sequences in the entire genome
What are cDNA libraries?
Comprised of expressed sequences by using RNA to make cDNA
