[MOD16A - BIOCHEM] Recombinant DNA Technology_2028

Technique used to combine DNA from different sources

Recombinant DNA Technology

A hybrid DNA molecule formed by combining DNA from different sources

Chimeric DNA

[T/F] Recombinant DNA technology can be used to correct genetic defects.

TRUE

Which enzyme cleaves DNA at specific palindromic sites?

Restriction enzyme (type II endonuclease)

How does a sticky end differ from a blunt end?

Sticky ends have unpaired overhangs; blunt ends are cut straight with no overhang

What determines how often a restriction enzyme cuts DNA?

The length of its recognition site (cut frequency = 4^n)

If BamHX is a 6-base cutter, how often will it cut a DNA sequence?

Once every 4096 bases

What is the cut frequency of a 6-base pair cutter like EcoRI?

Once every 4096 bases

Why are palindromic sequences important in restriction digestion?

Because restriction enzymes recognize and cut these symmetric sequences

What type of enzyme cuts DNA from the ends?

Exonuclease

What type of enzyme cuts DNA within the strand?

Endonuclease

A DNA fragment (gene or regulatory element) inserted into a vector for cloning or study.

Gene insert

A collection of cloned DNA fragments representing an organism's genome.

DNA library

Why is the cDNA library considered more specialized than the genomic library?

It only contains expressed genes (exons), excluding regulatory elements and introns

Why does cDNA library not contain introns?

Because it is reverse-transcribed from processed mRNA

Why is mRNA used to generate a cDNA library?

Because it contains only the expressed coding sequences (exons), allowing synthesis of cDNA that reflects functional genes

What is the role of an origin of replication in a vector?

Allows the inserted gene to be replicated within host cells

What are selectable markers used for in cloning vectors?

To identify cells that have taken up the recombinant DNA

What is the purpose of inserting DNA at the LacZ site?

To disrupt LacZ function, enabling blue-white selection of recombinants

Small, circular, double-stranded DNA molecules that replicate independently of host DNA

Plasmids

Why is the LacZ gene important in cloning?

It allows blue-white screening to identify recombinants

What does it mean if colonies are white on X-gal media?

LacZ is disrupted; likely contains DNA insert

What does it mean if colonies are blue on X-gal media?

LacZ gene is intact; no insert present

Which colonies should be eliminated in blue-white screening and how?

Host cells without plasmid; eliminate using ampicillin in the medium

[T/F] Colonies without plasmid will appear blue.

FALSE. They appear white due to absence of beta-galactosidase.

How do you eliminate non-plasmid colonies in blue-white screening?

Use X-gal medium with ampicillin to select only transformed cells

Why are plasmids with LacZ deletions used as host strains?

To enable alpha-complementation by the inserted LacZ portion in the vector

What happens when DNA is inserted into the ampicillin resistance gene?

It disrupts the gene, making the cell sensitive to ampicillin

If a chimeric plasmid has ampicillin resistance gene disrupted, what antibiotic should be used to select for transformants?

Tetracycline

[T/F] The presence of both ampicillin and tetracycline resistance indicates a recombinant plasmid.

FALSE. It indicates a plasmid without insert.

Vectors with promoters and Shine-Dalgarno sequences to drive gene expression

Expression vectors

What does PCR stand for?

Polymerase Chain Reaction

Who developed PCR and when?

Kary Mullis in 1983

What enzyme is used in PCR?

Heat-stable DNA polymerase (e.g., Taq)

What type of primers are used in PCR?

Short DNA primers flanking the region of interest

[T/F] RNA primers are used in PCR.

FALSE. PCR uses DNA primers, unlike natural replication.

PCR combined with reverse transcription to quantify RNA

RT-PCR

Quantitative PCR that amplifies and simultaneously quantifies DNA

Real-time PCR

How does real-time PCR quantify DNA?

It detects fluorescence during amplification. Amount of fluorescence is proportional to DNA amount

What nucleotide analog is used in Sanger sequencing to stop DNA synthesis?

2′,3′-dideoxynucleotide triphosphates

How are sequencing gels interpreted?

Read from bottom (shortest fragment, 5′) to top (longest, 3′) for the sequence

Why do dideoxynucleotides stop DNA polymerization in Sanger sequencing?

They lack a 3'-OH group needed to form phosphodiester bonds with incoming nucleotides

What does blotting detect?

Specific DNA, RNA, or proteins in complex samples

Southern blot detects what type of molecule?

DNA

Northern blot detects what type of molecule?

RNA

Western blot detects what type of molecule?

Protein

Labeled single-stranded DNA that hybridizes to a complementary target

DNA probe

What does ASO stand for?

Allele-Specific Oligonucleotide

What is the use of ASO probes?

Detect specific mutations in dot blots

How long are ASO probes typically?

15-20 nucleotides

Why are two probes used in ASO analysis for cystic fibrosis?

One for normal allele, one for mutated allele

Variation in DNA fragment lengths due to differences in restriction enzyme sites

Restriction Fragment Length Polymorphism (RFLP)

What causes loss of a restriction site in RFLP?

A point mutation in the recognition sequence that prevents the enzyme from binding or cutting

What causes the polymorphism in RFLP?

Mutations in restriction sites that prevent cutting

Describe the gel result of RFLP if one restriction site is mutated.

Fewer and longer fragments compared to normal

How is DNA visualized in RFLP after gel electrophoresis?

Radioactive probe binds to target DNA and shows bands on X-ray film

What is the principle behind RFLP pattern uniqueness between individuals?

Differences in restriction site mutations create unique fragment lengths

In RFLP, how would you interpret three bands in an individual?

They are heterozygous carriers (Aa)

Why is microarray more powerful than single-gene tests?

It allows simultaneous analysis of thousands of genes or mutations

What is each spot on a microarray chip?

A fixed DNA segment representing a gene or gene region

What type of sample is applied to a DNA microarray chip during analysis?

Labeled nucleic acid (target DNA or RNA) to be hybridized with immobilized probes

A researcher is working with a plasmid vector containing an intact LacZ gene and an ampicillin resistance gene. After inserting a DNA fragment into the LacZ region, the recombinant plasmid is introduced into E. coli. The bacteria are plated on X-gal + ampicillin media. What colony color and antibiotic resistance would confirm successful insertion?

White colonies that are ampicillin-resistant

A 6-year-old male presents with muscle weakness and delayed motor milestones. Genetic testing reveals a frameshift mutation in the dystrophin gene. Which inheritance pattern is most consistent with this case, and what is the likely diagnosis?

X-linked recessive; Duchenne Muscular Dystrophy

You are designing a probe to identify a point mutation in the CFTR gene known to cause cystic fibrosis. Which type of probe would be most appropriate, and what method should be used to test multiple family members quickly?

Allele-Specific Oligonucleotide (ASO); Dot blot

A researcher performs a Southern blot using DNA from two siblings. The restriction enzyme cuts DNA from sibling A into 3 fragments but from sibling B into only 2 fragments. What is the most likely explanation?

Sibling B has a mutation in a restriction site, preventing enzyme cleavage at that site (RFLP)

A male patient's pedigree shows no male-to-male transmission of a bleeding disorder, but several affected males are seen across generations through female carriers. What mode of inheritance is most likely, and what condition could explain this pattern?

X-linked recessive; Hemophilia A or B

Give three common examples of recombinant DNA pairings

Mouse and human DNA

Viral and bacterial DNA

Vector and target DNA

Differentiate genomic library from cDNA library

Genomic library: includes introns, exons, and regulatory sequences

cDNA library: includes only exons (from mRNA), no regulatory regions or introns

What are the key features of a good cloning vector?

Multiple restriction sites for DNA insertion

Origin of replication

Selectable marker genes (e.g., antibiotic resistance)

Enumerate types of cloning vectors with sizes

YACs - 200-500 kb

BACs - 100-200 kb

Cosmid - 45 kb

Phage - 15 kb

Plasmid - 5-10 kb

What two sequence elements are needed in an expression vector to allow protein production?

Promoter site

Shine-Dalgarno sequence

Enumerate uses of PCR

DNA fingerprinting

Antenatal diagnosis of gene mutations

Studying gene polymorphisms

What are the outputs of real-time PCR?

Absolute DNA copy numbers

Relative DNA expression normalized to input or control

Differentiate Maxam-Gilbert from Sanger sequencing

Maxam-Gilbert: chemical cleavage at specific bases

Sanger: chain termination using dideoxynucleotides

What is the general process of blotting across all techniques?

Separate molecules via electrophoresis

Transfer to membrane

Hybridize with a labeled probe

What is DNA microarray used for?

Detecting gene expression

Identifying mutations or gene variants

Gene mapping