Molecular biology methods and experimental manipulation Flashcards
(36 cards)
Restriction enzymes
Cut/cleave DNA often palindromic sections as they recognise specific sequences
Sticky ends
Parts left of when DNA is cleaved by an enzyme.
What sequence does enzyme Hipa1 find and where does it cut?
5-3’ GTT (CUT) AAC
What sequence does enzyme EcoR1 find and where does it cut?
5-3’ G. (cut). AATTC
What sequence does enzyme Hind111 find and where does it cut?
5-3’ A. (CUT). AGCTT
What sequence does enzyme Pst1 find and where does it cut?
5-3’ CTGCA. (CUT). G
What are Ligases?
Enzymes that stick compatible DNA back together.
If put in a test tube with sticky ends it will bind them back together with the use of ATP.
Globin Genes
Made in blood cells/red cells.
How is DNA cloned and amplified?
What is the most common?
In Microorganisms using Vectors (self-replicating genetic elements). Most common are those of E.coli Plasmids.
What are plasmids?
Double stranded circular DNA molecules that carry drug resistance genes, conferring resistance to certain antibiotics. Approx <20 kiloBase pairs (kb).
They contain an E.Coli origin of replication, when it copies DNA it also copies chromosomal sequence.
Also include gene of resistance (for example bla gene is ampicillin resistant).
2 outcomes of placing Ligase, DNA fragment and ATP in solution with plasmid and restriction enzyme?
Either plasmid joins back together to form original plasmid. Or accepts DNA fragment into plasmid.
What is a ligation mix?
When it is placed back into E.Coli what is it called?
ATP + ligase + DNA fragment + plasmid + restriction enzyme.
It is then put back into E.Coli.
Called transformation.
Agar Plates
Allow E.Coli to grow
Why would the E.coli cells fail to grow on ampicillin plates?
They did not accept the plasmid containing the ampicillin resistant gene.
Why would some plasmids not show their colour?
because the gene creating the blue would have been disrupted by accepting the DNA fragment.
Cell culture
Reproduces millions of new cells.
What are the 2 libraries of DNA?
Genomic libraries Complementary DNA (cDNA libraries)
What are Genomic libraries?
Purify genomic DNA and cleave with restriction enzyme = enormous number of DNA fragments, then cloned into (plasmid) E.Coli (or bacteria).Millions of E.Coli randomly encoding a different part of the human genome (as example was human DNA). Then find gene your interested in.
What are cDNA libraries?
Complementary DNA library generated from mRNA from tissue of interest.
Purified and then used as templates to make DNA copies.
Copies then used to generate library.
Reverse transcriptase
Enzyme used to make complementary DNA from mRNA.
Degrade mRNA with RNase and then synthesise a second DNA strand using DNA polymerase.
What are the difference between cDNA and genomic libraries?
cDNA library - Only contains copies of genes that are expressed in that tissue (only find globin expressing genes in blood cells). Only those sequences encoding proteins (exon sequences). They are smaller so easier to manipulate. Most useful for determining the amino acid structure of a protein.
Genomic - Span entire genome (intons, exons, promoter DNA and intergenic DNA). Helps determine structure of genome (which ones are next to each other). Used to isolate promoter and regulatory DNA fragments. Needed to explain why particular genes are expressed only in certain tissue.
What are exons and introns?
An exon is the portion of a gene that codes for amino acids. In the cells of plants and animals, most gene sequences are broken up by one or more DNA sequences called introns.
What is intergenic DNA
An Intergenic region is a stretch of DNA sequences located between genes. Intergenic regions are a subset of noncoding DNA. Occasionally some intergenic DNA acts to control genes nearby, but most of it has no currently known function.
What is promoter DNA?
In genetics, a promoter is a sequence of DNA to which proteins bind that initiate transcription of a single RNA from the DNA downstream of it.
